(Advances in Protein Chemistry and Structural Biology Volume 95) Donev, Rossen-Proteomics in Biomedicine and Pharmacology-Academic Press (2014)
(Advances in Protein Chemistry and Structural Biology Volume 95) Donev, Rossen-Proteomics in Biomedicine and Pharmacology-Academic Press (2014)
(Advances in Protein Chemistry and Structural Biology Volume 95) Donev, Rossen-Proteomics in Biomedicine and Pharmacology-Academic Press (2014)
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ISBN: 978-0-12-800453-1
ISSN: 1876-1623
14 15 16 17 10 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
Khaled Alawam
Forensic Medicine Department, Ministry of Interior, Kuwait City, Kuwait
Hossein Baharvand
Department of Developmental Biology, University of Science and Culture, and Department
of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute
for Stem Cell Biology and Technology, ACECR, Tehran, Iran
A. Elizabeth Bond
Institute of Mass Spectrometry, College of Medicine, Swansea University, Swansea, United
Kingdom
Christoph H. Borchers
University of Victoria—Genome British Columbia Proteomics Centre, and Department of
Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, Victoria,
British Columbia, Canada
Nicola Luigi Bragazzi
Nanobiotechnology and Biophysics Laboratories (NBL), Department of Experimental
Medicine (DIMES); Nanoworld Institute Fondazione ELBA Nicolini (FEN), Pradalunga,
Bergamo, and School of Public Health, Department of Health Sciences (DISSAL),
University of Genoa, Genoa, Italy
Juan Casado-Vela
Centro Nacional de Biotecnologı́a, Spanish National Research Council (CSIC), Madrid,
Spain
Ed Dudley
Institute of Mass Spectrometry, College of Medicine, Swansea University, Swansea, United
Kingdom
Octavio Luiz Franco
Centro de Análises Proteômicas e Bioquı́micas, Programa de Pós-Graduação em Ciências
Genômicas e Biotecnologia, Universidade Católica de Brası́lia, Brası́lia, Brazil
José Manuel Franco-Zorrilla
Centro Nacional de Biotecnologı́a, Spanish National Research Council (CSIC), Madrid,
Spain
Dustin C. Frost
School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA
Manuel Fuentes
Centro de Investigación del Cáncer/IBMCC (USAL/CSIC), IBSAL, Departamento de
Medicina, Unidad de Proteomics & Servicio General de Citometrı́a, University of
Salamanca, Salamanca, Spain
ix
x Contributors
Lingjun Li
School of Pharmacy, and Department of Chemistry, University of Wisconsin, Madison,
Wisconsin, USA
Claudio Nicolini
Nanobiotechnology and Biophysics Laboratories (NBL), Department of Experimental
Medicine (DIMES), University of Genoa, Genoa; Nanoworld Institute Fondazione ELBA
Nicolini (FEN), Pradalunga, Bergamo, Italy, and Biodesign Institute, Arizona State
University, Tempe, Arizona, USA
Eugenia Pechkova
Nanobiotechnology and Biophysics Laboratories (NBL), Department of Experimental
Medicine (DIMES), University of Genoa, Genoa, and Nanoworld Institute Fondazione
ELBA Nicolini (FEN), Pradalunga, Bergamo, Italy
Bernardo A. Petriz
Centro de Análises Proteômicas e Bioquı́micas, Programa de Pós-Graduação em Ciências
Genômicas e Biotecnologia, Universidade Católica de Brası́lia, Brası́lia, Brazil
Evgeniy V. Petrotchenko
University of Victoria—Genome British Columbia Proteomics Centre, Victoria, British
Columbia, Canada
Ghasem Hosseini Salekdeh
Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute
for Stem Cell Biology and Technology, ACECR, Tehran, and Department of Systems
Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran
Faezeh Shekari
Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute
for Stem Cell Biology and Technology, and Department of Developmental Biology,
University of Science and Culture, ACECR, Tehran, Iran
PREFACE
xi
xii Preface
Application of Cutting-Edge
Proteomics Technologies for
Elucidating Host–Bacteria
Interactions
Bernardo A. Petriz, Octavio Luiz Franco1
Centro de Análises Proteômicas e Bioquı́micas, Programa de Pós-Graduação em Ciências Genômicas e
Biotecnologia, Universidade Católica de Brası́lia, Brası́lia, Brazil
1
Corresponding author: e-mail address: [email protected]; [email protected]
Contents
1. Introduction 2
2. Classical Proteomics Strategies for Biomedical Research in General 2
2.1 Gel-based methods 4
3. Gel-Free Methods 5
3.1 Gel-free-labeling methods 6
3.2 Label-free and absolute quantification 8
4. New Proteomic Methods in Looking for Bacterial Pathogens 9
5. Proteomic Advances in Looking for Host Organisms 13
6. Prospects 17
References 18
Abstract
Advanced techniques and high-throughput protein analysis have led proteomics to
substantive progress in the understanding of bacterial–host interactions. Mass spec-
trometry (MS)-based proteomics have been a central methodology in the discovery
of new protein involved in the infectious process that leads to thousands of deaths
every year. The discovery of novel protein targets, together with de novo drug design,
improves the accuracy of early diagnosis, leading to improved new treatments.
MS-based proteomics has also been widely applied to structural biology, where prote-
omic investigation is being used to improve knowledge on the relationship between
protein sequence, structure, and function. Thus, the search for therapeutic targets for
infectious diseases using these cutting-edge technologies represents the new frontiers
for proteomics applications in biomedicine and pharmacology. In this review, the main
classical gel-based methods (2-DE, DIGE) are discussed, as well as the advances of gel-
free quantitative proteomic techniques, from metabolic and chemical labeling (SILAC,
iTRAQ, ICAT, 16O/18O, QconCAT) to nonlabeling (MS spectra counting and peak integra-
tion) strategies. Together, these proteomic methods are currently being used in the
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 1
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00001-4
2 Bernardo A. Petriz and Octavio Luiz Franco
quest for tailor-made pharmaceutical and biomedical research for bacterial control,
where advances in these analytical methods may represent greater improvements in
the treatment of a number of infectious diseases.
1. INTRODUCTION
In recent decades, infectious diseases caused by microorganisms have
become an increasing health problem. Bacterial infections caused by resistant
strains are of grave concern in numerous hospitals around the world, espe-
cially in elderly patients, those compromised by illness and those receiving
immunity-suppressant drugs (Grundmann et al., 2011). In this context, it is
essential to improve the understanding of infectious mechanisms and the
host response in order to develop drugs with potential activity against these
pathogens. To fill the manifold gaps that remain in our understanding of bac-
terial infectious processes, proteomics has been widely used (Cox et al.,
2012). In recent years, proteomic tools have accomplished significant
advances in the characterization of proteins involved in the mechanism of
infectious pathogens and also in the patient’s response (Lima et al., 2013).
In this context, this review focuses on proteomics tools used in the better
understanding of proteins involved in infectious processes in microorganism
and mammals, providing a broad overview of proteins possibly related to the
resistance process.
Thakur et al., 2008; Toepfer et al., 2013; Trombino et al., 2004; Walker,
Fullerton, & Buttrick, 2013).
Hence, biomedical and pharmacology fields have benefited from the
great advances in MS-based proteomics, fundamental for high-throughput
biomarker screening and development of novel pharmacologic strategies
(Berna et al., 2008; Thierolf et al., 2008; Vasudev et al., 2008; Yang
et al., 2011). In this review, the application of MS-based proteomics tools
and strategies in biomedical and pharmacologic fields will be addressed
for bacterial control and the treatment of a number of infectious diseases.
Section 1 focuses on the proteomic tools used for quantitative and qualitative
analysis followed by their application in the research of host–bacterial
interactions.
3. GEL-FREE METHODS
As mentioned before, a limited sample is a common situation in sev-
eral biomedical fields (e.g., rare cancer and invasive procedures), sometimes
becoming a restrictive issue for some proteomic techniques such as huge sets
of 2-DE gels, which usually require a high amount of sample. Therefore, the
option for gel-free methods is often applied as an alternative to gel-based
techniques, since a low amount of sample is required and because peptide
mixture is less complex to analyze compared to proteins (May et al.,
2011). The direct connection of LC to MS analyzers leads these methods
to be referred to as MS-based methods.
A typical LC-MS workflow begins with the protein sample being enzy-
matically digested (e.g., trypsin, chymotrypsin, Lys-C, Glu-C, Asp-N),
with the resultant peptide mixture being separated by 1D or multi-
dimensional chromatography (HPLC or UPLC, also performed in nano-
scale) depending on its complexity. Eluted peptides are then loaded
directly (on line) to electrospray ionization (ESI) for further MS analysis
(e.g., LC-ESI-MS, LC-ESI-MS/MS). After LC separation, digested pep-
tides may also be connected indirectly (off line) to MS by automated loading
of eluted fractions into MALDI steel plates for solid ionization process and
subsequent MS analysis (e.g., LC-MALDI-MS, LC-MALDI-MS/MS)
(Bodnar, Blackburn, Krise, & Moseley, 2003). After MS analysis, data on
peptide masses and fragmentation of ion masses are researched against a
6 Bernardo A. Petriz and Octavio Luiz Franco
SILAC is considered the technique that achieves the most accurate results.
Alternative metabolic labeling is also performed by 15N labeling method,
where all nitrogen atoms in peptides are labeled (DeSouza & Siu, 2013).
This method is preferred for quantifying autotrophic cells, thus being
more efficient in quantifying plant proteome (Oeljeklaus, Meyer, &
Warscheid, 2009).
rather than exclusive (Wu, Wang, Baek, & Shen, 2006). Figure 1.1 summa-
rizes the wide range of proteomic tools described in this review that are used
in biomedical and pharmacologic research.
Figure 1.2 Differential protein expression observed in pathogenic bacteria (left side),
human host (right side), and during the bacterial infection over human host (middle).
14 Bernardo A. Petriz and Octavio Luiz Franco
6. PROSPECTS
Although several different types of research have been presented here,
the use of proteomics in bacterial pathogens and host mammalian interac-
tions in order to prevent, cure, or simply understand some infectious diseases
is just beginning. At the moment, only a little information has been obtained
in various animals and cell models and less work has been done in humans.
For that, we must fill several gaps in knowledge and methodologies. It is
obvious that classical techniques have been extremely useful, including
2-DE MS, MS/MS, and label-MS methods, as previously described.
Nevertheless, such methods must be combined with others, including
immunoassays, electron microscopy, histological pathology, and infe-
ctology, transforming the task of understanding the host–pathogen interac-
tion into a multidisciplinary activity. Moreover, novel techniques and
approaches are extremely welcome in improving this information, and these
may include laser ablation ESI, for example (Kiss, Smith, Reschke, Powel, &
Heeren, 2013), which is a novel technique for MS imaging. This technique
allows lipids and small metabolites to be imaged in different samples such as
tissue sections and bacterial colonies without pretreatment. Moreover, laser
ablation ESI seems to be valuable also for the identification of proteins
directly from sample surfaces, and this could be extremely desirable for
experiments between bacteria and humans. This technique and others could
be interesting to map such interactions in real time.
18 Bernardo A. Petriz and Octavio Luiz Franco
REFERENCES
Aas, F. E., Egge-Jacobsen, W., Winther-Larsen, H. C., Lovold, C., Hitchen, P. G., Dell, A.,
et al. (2006). Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifi-
cations with phosphoethanolamine and phosphocholine requiring an enzyme structur-
ally related to lipopolysaccharide phosphoethanolamine transferases. The Journal of
Biological Chemistry, 281(38), 27712–27723.
Aebersold, R., & Mann, M. (2003). Mass spectrometry-based proteomics. Nature,
422(6928), 198–207.
Andersen, P., & Doherty, T. (2005). TB subunit vaccines—Putting the pieces together.
Microbes and Infection, 7, 911–921.
Anderson, N. L., & Anderson, N. G. (2002). The human plasma proteome: History, char-
acter, and diagnostic prospects. Molecular & Cellular Proteomics, 1(11), 845–867 (Review).
Anonsen, J. H., Egge-Jacobsen, W., Aas, F. E., Borud, B., Koomey, M., & Vik, A. (2012).
Novel protein substrates of the phospho-form modification system in Neisseria
gonorrhoeae and their connection to O-linked protein glycosylation. Infection and Immu-
nity, 80(1), 22–30.
Ansong, C., Wu, S., Meng, D., Liu, X., Brewer, H. M., Deatherage Kaiser, B. L., et al.
(2013). Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella
Typhimurium in response to infection-like conditions. Proceedings of the National Academy
of Sciences of the United States of America, 110(25), 10153–10158.
Austin, R. J., Chang, D. K., Holstein, C. A., Lee, L. W., Risler, J., Wang, J. H., et al. (2012).
IQcat: Multiplexed protein quantification by isoelectric QconCAT. Proteomics, 12(13),
2078–2083.
Banks, R. E., Dunn, M. J., Hochstrasser, D. F., Sanchez, J. C., Blackstock, W., Pappin, D. J.,
et al. (2000). Proteomics: New perspectives, new biomedical opportunities. Lancet,
356(9243), 1749–1756.
Berna, M., Ott, L., Engle, S., Watson, D., Solter, P., & Ackermann, B. (2008). Quantifica-
tion of NTproBNP in rat serum using immunoprecipitation and LC/MS/MS:
A biomarker of drug-induced cardiac hypertrophy. Analytical Chemistry, 80(3), 561–566.
Bodnar, W. M., Blackburn, R. K., Krise, J. M., & Moseley, M. A. (2003). Exploiting the
complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased
proteome coverage. Journal of the American Society for Mass Spectrometry, 14(9), 971–979.
Bougnoux, A. C., & Solassol, J. (2013). The contribution of proteomics to the identification
of biomarkers for cutaneous malignant melanoma. Clinical Biochemistry, 46(6), 518–523.
Castagna, A., Polati, R., Bossi, A. M., & Girelli, D. (2012). Monocyte/macrophage prote-
omics: Recent findings and biomedical applications. Expert Review of Proteomics, 9(2),
201–215.
Cutting-Edge Proteomics Technologies for Elucidating Host–Bacteria Interactions 19
Cha, I. S., Kwon, J., Park, S. H., Nho, S. W., Jang, H. B., Park, S. B., et al. (2012).
Kidney proteome responses in the teleost fish Paralichthys olivaceus indicate a putative
immune response against Streptococcus parauberis. Journal of Proteomics, 75(17),
5166–5175.
Chang, C. J., Lin, J. H., Chang, K. C., Lai, M. J., Rohini, R., & Hu, A. (2013). Diagnosis of
beta-lactam resistance in Acinetobacter baumannii using shotgun proteomics and
LC-nano-electrospray ionization ion trap mass spectrometry. Analytical Chemistry,
85(5), 2802–2808.
Charro, N., Hood, B. L., Faria, D., Pacheco, P., Azevedo, P., Lopes, C., et al. (2011). Serum
proteomics signature of cystic fibrosis patients: A complementary 2-DE and LC-MS/MS
approach. Journal of Proteomics, 74(1), 110–126.
Chopra, S., Ramkissoon, K., & Anderson, D. C. (2013). A systematic quantitative proteomic
examination of multidrug resistance in Acinetobacter baumannii. Journal of Proteomics, 84,
17–39.
Coler, R. N., Dillon, D. C., Skeiky, Y. A., Kahn, M., Orme, I. M., Lobet, Y., et al. (2009).
Identification of Mycobacterium tuberculosis vaccine candidates using human CD4 +
T-cells expression cloning. Vaccine, 27(2), 223–233.
Collado-Romero, M., Martins, R. P., Arce, C., Moreno, A., Lucena, C., Carvajal, A., et al.
(2012). An in vivo proteomic study of the interaction between Salmonella Typhimurium
and porcine ileum mucosa. Journal of Proteomics, 75(7), 2015–2026.
Cox, G., Thompson, G. S., Jenkins, H. T., Peske, F., Savelsbergh, A., Rodnina, M. V., et al.
(2012). Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic
fusidic acid. Proceedings of the National Academy of Sciences of the United States of America,
109(6), 2102–2107.
Cravatt, B. F., Simon, G. M., & Yates, J. R., III (2007). The biological impact of mass-
spectrometry-based proteomics. Nature, 450(7172), 991–1000.
De Masi, R., Pasca, S., Scarpello, R., Idolo, A., & De Donno, A. (2013). The clinical poten-
tial of blood-proteomics in multiple sclerosis. BMC Neurology, 13, 45.
Depke, M., Surmann, K., Hildebrandt, P., Jehmlich, N., Michalik, S., Stanca, S. E., et al.
(2014). Labeling of the pathogenic bacterium Staphylococcus aureus with gold or ferric
oxide-core nanoparticles highlights new capabilities for investigation of host-pathogen
interactions. Cytometry. Part A, 85, 140–150.
DeSouza, L. V., & Siu, K. W. (2013). Mass spectrometry-based quantification. Clinical Bio-
chemistry, 46(6), 421–431.
Domon, B., & Aebersold, R. (2006). Mass spectrometry and protein analysis. Science,
312(5771), 212–217.
Elschenbroich, S., Ignatchenko, V., Sharma, P., Schmitt-Ulms, G., Gramolini, A. O., &
Kislinger, T. (2009). Peptide separations by on-line MudPIT compared to isoelectric
focusing in an off-gel format: Application to a membrane-enriched fraction from
C2C12 mouse skeletal muscle cells. Journal of Proteome Research, 8(10), 4860–4869.
Eng, J. K., Jahan, T. A., & Hoopmann, M. R. (2013). Comet: An open-source MS/MS
sequence database search tool. Proteomics, 13(1), 22–24.
Fonslow, B. R., Carvalho, P. C., Academia, K., Freeby, S., Xu, T., Nakorchevsky, A., et al.
(2011). Improvements in proteomic metrics of low abundance proteins through prote-
ome equalization using ProteoMiner prior to MudPIT. Journal of Proteome Research,
10(8), 3690–3700.
Fonslow, B. R., Niessen, S. M., Singh, M., Wong, C. C., Xu, T., Carvalho, P. C., et al.
(2012). Single-step inline hydroxyapatite enrichment facilitates identification and
quantitation of phosphopeptides from mass-limited proteomes with MudPIT. Journal
of Proteome Research, 11(5), 2697–2709.
Franzel, B., & Wolters, D. A. (2011). Advanced MudPIT as a next step toward high prote-
ome coverage. Proteomics, 11(18), 3651–3656.
20 Bernardo A. Petriz and Octavio Luiz Franco
Frohlich, T., Helmstetter, D., Zobawa, M., Crecelius, A. C., Arzberger, T.,
Kretzschmar, H. A., et al. (2006). Analysis of the HUPO Brain Proteome reference sam-
ples using 2-D DIGE and 2-D LC-MS/MS. Proteomics, 6(18), 4950–4966.
Fu, Y. R., Yi, Z. J., Guan, S. Z., Zhang, S. Y., & Li, M. (2012). Proteomic analysis of sputum
in patients with active pulmonary tuberculosis. Clinical Microbiology and Infection, 18(12),
1241–1247.
Gault, J., Malosse, C., Dumenil, G., & Chamot-Rooke, J. (2013). A combined mass spec-
trometry strategy for complete posttranslational modification mapping of Neisseria
meningitidis major pilin. Journal of Mass Spectrometry, 48(11), 1199–1206.
Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., & Gygi, S. P. (2003).
Absolute quantification of proteins and phosphoproteins from cell lysates by tandem
MS. Proceedings of the National Academy of Sciences of the United States of America,
100(12), 6940–6945.
Ghafourian, S., Sekawi, Z., Raftari, M., & Ali, M. S. (2013). Application of proteomics in lab
diagnosis. Clinical Laboratory, 59(5–6), 465–474.
Giltner, C. L., Habash, M., & Burrows, L. L. (2010). Pseudomonas aeruginosa minor pilins
are incorporated into type IV pili. Journal of Molecular Biology, 398(3), 444–461.
Gonzalez-Begne, M., Lu, B., Han, X., Hagen, F. K., Hand, A. R., Melvin, J. E., et al. (2009).
Proteomic analysis of human parotid gland exosomes by multidimensional protein iden-
tification technology (MudPIT). Journal of Proteome Research, 8(3), 1304–1314.
Gonzalez-Begne, M., Lu, B., Liao, L., Xu, T., Bedi, G., Melvin, J. E., et al. (2011). Char-
acterization of the human submandibular/sublingual saliva glycoproteome using lectin
affinity chromatography coupled to multidimensional protein identification technology.
Journal of Proteome Research, 10(11), 5031–5046.
Gorg, A., Weiss, W., & Dunn, M. J. (2004). Current two-dimensional electrophoresis tech-
nology for proteomics. Proteomics, 4(12), 3665–3685.
Griffin, T. J., Gygi, S. P., Rist, B., Aebersold, R., Loboda, A., Jilkine, A., et al. (2001). Quan-
titative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer.
Analytical Chemistry, 73(5), 978–986.
Grundmann, H., Klugman, K. P., Walsh, T., Ramon-Pardo, P., Sigauque, B., Khan, W.,
et al. (2011). A framework for global surveillance of antibiotic resistance. Drug Resistance
Updates, 14(2), 79–87.
Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., & Aebersold, R. (1999). Quan-
titative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Bio-
technology, 17(10), 994–999.
Harrison, P. M., Kumar, A., Lang, N., Snyder, M., & Gerstein, M. (2002). A question of size:
The eukaryotic proteome and the problems in defining it. Nucleic Acids Research, 30(5),
1083–1090.
Hoyos-Mallecot, Y., Cabrera-Alvargonzalez, J. J., Miranda-Casas, C., Rojo-Martin, M. D.,
Liebana-Martos, C., & Navarro-Mari, J. M. (2014). MALDI-TOF MS, a useful instru-
ment for differentiating metallo-beta-lactamases in Enterobacteriaceae and Pseudomonas
spp. Letters in Applied Microbiology, 58, 325–329.
Julka, S., & Regnier, F. E. (2005). Recent advancements in differential proteomics based on
stable isotope coding. Briefings in Functional Genomics & Proteomics, 4(2), 158–177.
Jung, J. S., Eberl, T., Sparbier, K., Lange, C., Kostrzewa, M., Schubert, S., et al. (2013).
Rapid detection of antibiotic resistance based on mass spectrometry and stable isotopes.
European Journal of Clinical Microbiology & Infectious Diseases. In press.
Jungblut, P. R., Schiele, F., Zimny-Arndt, U., Ackermann, R., Schmid, M., Lange, S., et al.
(2010). Helicobacter pylori proteomics by 2-DE/MS, 1-DE-LC/MS and functional data
mining. Proteomics, 10(2), 182–193.
Kiss, A., Smith, D. F., Reschke, B. R., Powel, M. J., & Heeren, R. M. (2013). Top-down
mass spectrometry imaging of intact proteins by LAESI FT-ICR MS. Proteomics. In press.
Cutting-Edge Proteomics Technologies for Elucidating Host–Bacteria Interactions 21
Kolarova, M., Garcia-Sierra, F., Bartos, A., Ricny, J., & Ripova, D. (2012). Structure and
pathology of tau protein in Alzheimer disease. International Journal of Alzheimer’s Disease,
2012, 731526.
Konvalinka, A., Scholey, J. W., & Diamandis, E. P. (2012). Searching for new biomarkers of
renal diseases through proteomics. Clinical Chemistry, 58(2), 353–365.
Kunnath-Velayudhan, S., & Porcelli, S. A. (2013). Recent advances in defining the immu-
noproteome of Mycobacterium tuberculosis. Frontiers in Immunology, 4, 335.
Kunnath-Velayudhan, S., Salamon, H., Wang, H. Y., Davidow, A. L., Molina, D. M.,
Huynh, V. T., et al. (2010). Dynamic antibody responses to the Mycobacterium
tuberculosis proteome. Proceedings of the National Academy of Sciences of the United States
of America, 107(33), 14703–14708.
Lee, S. W., Kim, I. J., Jeong, B. Y., Choi, M. H., Kim, J. Y., Kwon, K. H., et al. (2012). Use
of MDLC-DIGE and LC-MS/MS to identify serum biomarkers for complete remission
in patients with acute myeloid leukemia. Electrophoresis, 33(12), 1863–1872.
Lee, J. C., Lee, E. J., Lee, J. H., Jun, S. H., Choi, C. W., Kim, S. I., et al. (2012). Klebsiella
pneumoniae secretes outer membrane vesicles that induce the innate immune response.
FEMS Microbiology Letters, 331(1), 17–24.
Li, Y., Zeng, J., Shi, J., Wang, M., Rao, M., Xue, C., et al. (2010). A proteome-scale iden-
tification of novel antigenic proteins in Mycobacterium tuberculosis toward diagnostic
and vaccine development. Journal of Proteome Research, 9(9), 4812–4822.
Lima, T. B., Pinto, M. F., Ribeiro, S. M., de Lima, L. A., Viana, J. C., Gomes Junior, N.,
et al. (2013). Bacterial resistance mechanism: What proteomics can elucidate. The
FASEB Journal, 27(4), 1291–1303.
Lindestam Arlehamn, C. S., Gerasimova, A., Mele, F., Henderson, R., Swann, J.,
Greenbaum, J. A., et al. (2013). Memory T cells in latent Mycobacterium tuberculosis
infection are directed against three antigenic islands and largely contained in a CXCR3
+CCR6 + Th1 subset. PLoS Pathogens, 9(1), e1003130.
Lu, Q., Bai, J., Zhang, L., Liu, J., Jiang, Z., Michal, J. J., et al. (2012). Two-dimensional
liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative
and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles
of pulmonary alveolar macrophages infected with porcine reproductive and respiratory
syndrome virus. Journal of Proteome Research, 11(5), 2890–2903.
Mahdavi, A., Szychowski, J., Ngo, J. T., Sweredoski, M. J., Graham, R. L., Hess, S., et al.
(2014). Identification of secreted bacterial proteins by noncanonical amino acid tagging.
Proceedings of the National Academy of Sciences of the United States of America, 111, 433–438.
Maria-Neto, S., Candido Ede, S., Rodrigues, D. R., de Sousa, D. A., da Silva, E. M., de
Moraes, L. M., et al. (2012). Deciphering the magainin resistance process of Escherichia
coli strains in light of the cytosolic proteome. Antimicrobial Agents and Chemotherapy,
56(4), 1714–1724.
Martins, R. P., Collado-Romero, M., Martinez-Gomariz, M., Carvajal, A., Gil, C.,
Lucena, C., et al. (2012). Proteomic analysis of porcine mesenteric lymph-nodes after
Salmonella typhimurium infection. Journal of Proteomics, 75(14), 4457–4470.
May, C., Brosseron, F., Chartowski, P., Schumbrutzki, C., Schoenebeck, B., & Marcus, K.
(2011). Instruments and methods in proteomics. Methods in Molecular Biology, 696, 3–26.
Melo, J., Schrama, D., Andrew, P. W., & Faleiro, M. L. (2013). Proteomic analysis shows
that individual Listeria monocytogenes strains use different strategies in response to gas-
tric stress. Foodborne Pathogens and Disease, 10(2), 107–119.
Melo, J., Schrama, D., Hussey, S., Andrew, P. W., & Faleiro, M. L. (2013). Listeria
monocytogenes dairy isolates show a different proteome response to sequential expo-
sure to gastric and intestinal fluids. International Journal of Food Microbiology, 163(2–3),
51–63.
Minden, J. S. (2012). DIGE: Past and future. Methods in Molecular Biology, 854, 3–8 (Review).
22 Bernardo A. Petriz and Octavio Luiz Franco
Mitulovic, G., & Mechtler, K. (2006). HPLC techniques for proteomics analysis—A short
overview of latest developments. Briefings in Functional Genomics & Proteomics, 5(4),
249–260.
Nagele, E., Vollmer, M., Horth, P., & Vad, C. (2004). 2D-LC/MS techniques for the iden-
tification of proteins in highly complex mixtures. Expert Review of Proteomics, 1(1), 37–46
(Review).
Oeljeklaus, S., Meyer, H. E., & Warscheid, B. (2009). Advancements in plant proteomics
using quantitative mass spectrometry. Journal of Proteomics, 72(3), 545–554.
Old, W. M., Meyer-Arendt, K., Aveline-Wolf, L., Pierce, K. G., Mendoza, A.,
Sevinsky, J. R., et al. (2005). Comparison of label-free methods for quantifying human
proteins by shotgun proteomics. Molecular & Cellular Proteomics, 4(10), 1487–1502 (Com-
parative Study).
Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., et al.
(2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and
accurate approach to expression proteomics. Molecular & Cellular Proteomics, 1(5),
376–386.
Oswald, S., Groer, C., Drozdzik, M., & Siegmund, W. (2013). Mass spectrometry-based
targeted proteomics as a tool to elucidate the expression and function of intestinal drug
transporters. The AAPS Journal, 15(4), 1128–1140.
Pajuaba, A. C., Silva, D. A., Almeida, K. C., Cunha-Junior, J. P., Pirovani, C. P.,
Camillo, L. R., et al. (2012). Immunoproteomics of Brucella abortus reveals differential
antibody profiles between S19-vaccinated and naturally infected cattle. Proteomics, 12(6),
820–831.
Papasergi, S., Galbo, R., Lanza-Cariccio, V., Domina, M., Signorino, G., Biondo, C., et al.
(2013). Analysis of the Streptococcus agalactiae exoproteome. Journal of Proteomics, 89,
154–164.
Parguina, A. F., Rosa, I., & Garcia, A. (2012). Proteomics applied to the study of platelet-
related diseases: Aiding the discovery of novel platelet biomarkers and drug targets. Jour-
nal of Proteomics, 76(Spec No), 275–286.
Perkins, D. N., Pappin, D. J., Creasy, D. M., & Cottrell, J. S. (1999). Probability-based pro-
tein identification by searching sequence databases using mass spectrometry data.
Electrophoresis, 20(18), 3551–3567.
Petriz, B. A., Gomes, C. P., Rocha, L. A., Rezende, T. M., & Franco, O. L. (2012). Pro-
teomics applied to exercise physiology: A cutting-edge technology. Journal of Cellular
Physiology, 227, 885–898.
Pfortner, H., Wagner, J., Surmann, K., Hildebrandt, P., Ernst, S., Bernhardt, J., et al. (2013).
A proteomics workflow for quantitative and time-resolved analysis of adaptation reac-
tions of internalized bacteria. Methods, 61(3), 244–250.
Raggiaschi, R., Lorenzetto, C., Diodato, E., Caricasole, A., Gotta, S., & Terstappen, G. C.
(2006). Detection of phosphorylation patterns in rat cortical neurons by combining
phosphatase treatment and DIGE technology. Proteomics, 6(3), 748–756.
Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., et al.
(2004). Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-
reactive isobaric tagging reagents. Molecular & Cellular Proteomics, 3(12), 1154–1169.
Russell, M. R., Achour, B., McKenzie, E. A., Lopez, R., Harwood, M. D., Rostami-
Hodjegan, A., et al. (2013). Alternative fusion protein strategies to express recalcitrant
QconCAT proteins for quantitative proteomics of human drug metabolizing enzymes
and transporters. Journal of Proteome Research, 12(12), 5934–5942.
Scherp, P., Ku, G., Coleman, L., & Kheterpal, I. (2011). Gel-based and gel-free proteomic
technologies. Methods in Molecular Biology, 702, 163–190.
Schmidt, A., Kellermann, J., & Lottspeich, F. (2005). A novel strategy for quantitative pro-
teomics using isotope-coded protein labels. Proteomics, 5(1), 4–15 (Comparative Study).
Cutting-Edge Proteomics Technologies for Elucidating Host–Bacteria Interactions 23
Seibel, J., Konig, S., Gohler, A., Doose, S., Memmel, E., Bertleff, N., et al. (2013). Inves-
tigating infection processes with a workflow from organic chemistry to biophysics: The
combination of metabolic glycoengineering, super-resolution fluorescence imaging and
proteomics. Expert Review of Proteomics, 10(1), 25–31.
Selvaraju, S., & El Rassi, Z. (2011). Reduction of protein concentration range difference
followed by multicolumn fractionation prior to 2-DE and LC-MS/MS profiling of
serum proteins. Electrophoresis, 32(6–7), 674–685.
Sharma, V., Eng, J. K., Maccoss, M. J., & Riffle, M. (2012). A mass spectrometry proteomics
data management platform. Molecular & Cellular Proteomics, 11(9), 824–831.
Silva, J. C., Denny, R., Dorschel, C. A., Gorenstein, M., Kass, I. J., Li, G. Z., et al. (2005).
Quantitative proteomic analysis by accurate mass retention time pairs. Analytical Chem-
istry, 77(7), 2187–2200.
Silva, O. N., Mulder, K. C., Barbosa, A. E., Otero-Gonzalez, A. J., Lopez-Abarrategui, C.,
Rezende, T. M., et al. (2011). Exploring the pharmacological potential of promiscuous
host-defense peptides: From natural screenings to biotechnological applications. Frontiers
in Microbiology, 2, 232.
Tang, H. Y., Beer, L. A., & Speicher, D. W. (2011). In-depth analysis of a plasma or serum
proteome using a 4D protein profiling method. Methods in Molecular Biology, 728, 47–67.
Thakur, A., Siedlak, S. L., James, S. L., Bonda, D. J., Rao, A., Webber, K. M., et al. (2008).
Retinoblastoma protein phosphorylation at multiple sites is associated with neurofibril-
lary pathology in Alzheimer disease. International Journal of Clinical and Experimental
Pathology, 1(2), 134–146.
Thierolf, M., Hagmann, M. L., Pfeffer, M., Berntenis, N., Wild, N., Roessler, M., et al.
(2008). Towards a comprehensive proteome of normal and malignant human colon tis-
sue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as poten-
tial cancer biomarker. Proteomics Clinical Applications, 2(1), 11–22.
Thompson, A., Schafer, J., Kuhn, K., Kienle, S., Schwarz, J., Schmidt, G., et al. (2003). Tan-
dem mass tags: A novel quantification strategy for comparative analysis of complex pro-
tein mixtures by MS/MS. Analytical Chemistry, 75(8), 1895–1904.
Toepfer, C., Caorsi, V., Kampourakis, T., Sikkel, M. B., West, T. G., Leung, M. C., et al.
(2013). Myosin regulatory light chain (RLC) phosphorylation change as a modulator of
cardiac muscle contraction in disease. The Journal of Biological Chemistry, 288(19),
13446–13454.
Trombino, S., Bisio, A., Catassi, A., Cesario, A., Falugi, C., & Russo, P. (2004). Role of the
non-neuronal human cholinergic system in lung cancer and mesothelioma: Possibility of
new therapeutic strategies. Current Medicinal Chemistry Anti-Cancer Agents, 4(6), 535–542.
Unlu, M., Morgan, M. E., & Minden, J. S. (1997). Difference gel electrophoresis: A single gel
method for detecting changes in protein extracts. Electrophoresis, 18(11), 2071–2077.
Varnum, S. M., Webb-Robertson, B. J., Pounds, J. G., Moore, R. J., Smith, R. D.,
Frevert, C. W., et al. (2012). Proteomic analysis of bronchoalveolar lavage fluid proteins
from mice infected with Francisella tularensis ssp. novicida. Journal of Proteome Research,
11(7), 3690–3703.
Vasudev, N. S., Ferguson, R. E., Cairns, D. A., Stanley, A. J., Selby, P. J., & Banks, R. E.
(2008). Serum biomarker discovery in renal cancer using 2-DE and prefractionation by
immunodepletion and isoelectric focusing; increasing coverage or more of the same?
Proteomics, 8(23–24), 5074–5085.
Walker, L. A., Fullerton, D. A., & Buttrick, P. M. (2013). Contractile protein phosphory-
lation predicts human heart disease phenotypes. American Journal of Physiology. Heart and
Circulatory Physiology, 304(12), H1644–H1650.
Wang, J., Chen, C., Xie, P., Pan, Y., Tan, Y., & Tang, L. (2014). Proteomic analysis and
immune properties of exosomes released by macrophages infected with Mycobacterium
avium. Microbes and Infection, 16, 283–291.
24 Bernardo A. Petriz and Octavio Luiz Franco
Weeks, M. E. (2010). Urinary proteome profiling using 2D-DIGE and LC-MS/MS. Methods
in Molecular Biology, 658, 293–309.
Winnik, W. M., Dekroon, R. M., Jeong, J. S., Mocanu, M., Robinette, J. B., Osorio, C.,
et al. (2012). Analysis of proteins using DIGE and MALDI mass spectrometry. Methods in
Molecular Biology, 854, 47–66.
Wittmann-Liebold, B., Graack, H. R., & Pohl, T. (2006). Two-dimensional gel electropho-
resis as tool for proteomics studies in combination with protein identification by mass
spectrometry. Proteomics, 6(17), 4688–4703.
Wu, J., Shakey, Q., Liu, W., Schuller, A., & Follettie, M. T. (2007). Global profiling of phos-
phopeptides by titania affinity enrichment. Journal of Proteome Research, 6(12), 4684–4689
(Validation Studies).
Wu, W. W., Wang, G., Baek, S. J., & Shen, R. F. (2006). Comparative study of three pro-
teomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI
TOF/TOF. Journal of Proteome Research, 5(3), 651–658.
Yang, N., Feng, S., Shedden, K., Xie, X., Liu, Y., Rosser, C. J., et al. (2011). Urinary gly-
coprotein biomarker discovery for bladder cancer detection using LC/MS-MS and label-
free quantification. Clinical Cancer Research, 17(10), 3349–3359.
Zhao, Y., & Jensen, O. N. (2009). Modification-specific proteomics: Strategies for charac-
terization of post-translational modifications using enrichment techniques. Proteomics,
9(20), 4632–4641.
CHAPTER TWO
Phosphoproteomic Techniques
and Applications
Ed Dudley1, A. Elizabeth Bond
Institute of Mass Spectrometry, College of Medicine, Swansea University, Swansea, United Kingdom
1
Corresponding author: e-mail address: [email protected]
Contents
1. Introduction 25
2. Phosphoproteomic Methodologies 32
2.1 Phosphopeptide enrichment 33
2.2 Peptide separation by HPLC 38
2.3 MS analysis 42
3. Applications of Phosphoproteomics in Biomedicine 45
3.1 Applications in cancer research 45
3.2 Applications in stem cell research 53
3.3 Applications in cardiac research 55
3.4 Applications in immunity research 58
4. Discussion 59
References 60
Abstract
Phosphoproteomic analysis seeks to determine the overall level of protein phosphor-
ylation, as a result of kinase and phosphatase activity, and determine the identity of pro-
teins which are phosphorylated and the amino acid residues which hold the phosphate
group. The methodologies available have improved with increased research efforts;
however, the most commonly followed procedure is to enrich for phosphoproteins
or peptides and undertake tandem mass spectrometric analysis focusing on specific sig-
nature losses which represent phosphopeptides. There have been many advances in
this area and these are detailed both in relation to available protocols for phospho-
proteomic analysis and to the widening range of biomedical fields in which such
approaches are being commonly applied.
1. INTRODUCTION
High-throughput analysis has allowed for the study of changes in the
biological functions of cells at a variety of levels—ranging from the study of
differential gene expression, transcriptomic variations, alterations in the
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 25
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00002-6
26 Ed Dudley and A. Elizabeth Bond
protein complement at the cellular level, and further study of the biochem-
ical effect of these changes in the metabolite complement presented. The
technologies required to undertake these individual analyses and combina-
tions of them have continually developed over the last decade and are con-
stantly improving in relation to their throughput, reduction in cost of
analysis, the robustness of the data produced by the analyses, and the bioin-
formatics software solutions available to interpret the increasingly large data
sets produced. The combination of the different analyses aims to fully inter-
rogate the underlying cause behind a change in a cell’s behavior, be it trans-
formation into a tumor, differentiation into different cell types, or the
production of a diagnostically or prognostically useful biomarker of a specific
disorder or disease. Generally, the genome represents the overall comple-
ment of what may be produced by a cell while the transcriptome defines
which of these elements is being actively utilized at any given time point.
The proteome therefore is an illustration of which transcripts are being uti-
lized at the protein level, and the metabolome can be studied in order to
demonstrate the biochemical consequences of the previous analyses. In this
manner, combining such data sets allows for a fully developed image of how
different cells, tissues, or biological fluids may differ under different circum-
stances and this has a number of benefits in biomedical research and
pharmacology.
Phosphoproteomics represents a subdivision of proteomic analysis con-
cerned with the study of the phosphorylation status of proteins within a
biological sample (or comparisons between biological samples). Before dis-
cussing how protein phosphorylation can be monitored in a global manner,
it is worth considering the process of protein phosphorylation and its impli-
cations for the cell. The process of phosphorylation is performed by the
kinase enzymes having different specificities for the target proteins which
become phosphorylated and also the site of the phosphorylation. Com-
monly, phosphorylation occurs as a downstream effect of an external cell
receptor protein interacting with its associated ligand (commonly a hormone
or other circulating signaling molecule). Receptor activation causes a signal
transduction cascade to be initiated which can often either activate a tyrosine
kinase activity within the cytoplasmic section of the receptor protein or acti-
vate cytoplasmic protein kinases via the biosynthesis of secondary messen-
gers such as the cyclic nucleotides. Once activated, the kinase enzymes
utilize adenosine triphosphate (ATP) to add a phosphate residue to a specific
amino acid within the target protein structure. In mammalian systems, only
three amino acid residues are available for phosphorylation, these being
Phosphoproteomic Analysis in Biomedicine 27
threonine, tyrosine, and serine. The act of adding a phosphate group to the
amino acid residue has a number of consequences for the protein phosphor-
ylated. The addition of a phosphate residue to the amino acid sequence pro-
vides for two additional potential negative charges (arising from dissociated
oxygen atoms attached covalently to the phosphate itself ) and these are
therefore available to either disrupt existing electrostatic interactions or pro-
vide new interactions between sections of the protein. These oxygens and
hydroxyl components can also create new hydrogen bonds within the struc-
ture, and therefore conformational change of the protein’s overall structure
can occur due to the minor act of phosphorylation. Finally, the phosphate
residue itself is derived from the hydrolysis of ATP and this hydrolysis reac-
tion is highly exergonic and therefore provides a significant release of
energy. While approximately half of this energy is required to add the phos-
phate residue to the protein, the other half can be conserved by the protein
and/or the reactions which the protein then undertakes. As a result of these
factors, the simple act of phosphorylation can lead to a significant change in
the proteins activity—usually by bringing about conformational change
within the proteins overall structure which can lead to increased or
decreased activity of the protein and different kinetic parameters, such as
Km and Vmax, are usually exhibited by proteins in their different conforma-
tional forms. The common signal transduction systems that result in differ-
ential protein phosphorylation vary in their net result in different cells
allowing different mammalian organs to respond differently to the same cir-
culating “signal.” These systems react rapidly due to a number of factors;
first, the rate-limiting step in the phosphorylation reaction itself is the con-
centration of the phosphate-donating substrate, ATP. However, as ATP is
utilized as the cell’s major energy providing metabolite, the intercellular
levels of ATP are consistently maintained at a high concentration and this
ensures that the amount of ATP within a cell never becomes a rate-limiting
step in the phosphorylation response to a stimuli. Furthermore, the signal
transduction system by which a cell responds via differential protein phos-
phorylation to a change in hormonal levels or similar stimulus exhibits an
amplification cascade regarding the steps involved in eventual protein phos-
phorylation. For example, a single binding event of a single hormone at a
single extracellular receptor will activate that one receptor, the activated
receptor can then activate a single adenylate cyclase enzyme (as an example).
This single adenylate cyclase enzyme can then produce a large number
of cyclic adenosine monophosphate (cAMP) secondary messenger metabo-
lites inside the cell. Each secondary messenger may activate a single kinase
28 Ed Dudley and A. Elizabeth Bond
(such as protein kinase A) and this kinase in turn may activate multiple fur-
ther kinases. At each step where multiple end products are produced, the
single binding event is in effect amplified and therefore a single binding
can cause the eventual altered phosphorylation of a significant number of
individual proteins. Therefore, the response of the cell is rapid and substan-
tial for any particular binding event. The phosphorylation of proteins in this
manner is a reversible process and the removal of the phosphate from any
particular target protein is undertaken by a separate series of enzymes
referred to as phosphatases. Therefore, the overall protein phosphorylation
status of a cell is controlled by the activity of the kinase and phosphatase
enzymes which respond to stimuli which is external to the cell allowing
for coordinated regulation of cellular activity within organisms. The ability
of different cells to respond differently to similar stimuli is essential for dif-
ferent organs within mammalian systems to act in a relevant manner to any
given change (usually represented at the molecular level by an altered level of
a specific hormone released as a response by the given change). This spec-
ificity relies on a number of factors including whether or not the particular
cell produces an extracellular receptor for the particular hormone in ques-
tion and the isoform of the secondary messenger producing protein that is
produced by the cell in question. Different cells produce different isoforms
of the cyclase enzymes that produce the secondary messenger and also the
phosphodiesterases which catabolize the cyclic nucleotide secondary mes-
sengers. These different isoforms affect how the cell responds and how long
the signal that is being transduced is maintained. This variation in the
enzymes in different cells also allows for different therapeutic interventions
to be produced which effect selectively one specific isoform and therefore
only bring about an intervention in a specific target organ or cell without
effecting other tissues. A key example of this is the drug Viagra
(Sildenafil), which acts as an inhibitor of the type 5 class of cyclic guanosine
monophosphate (cGMP) phosphodiesterases. The inhibition of this enzyme
results in elevated cGMP levels being maintained most prominently in the
corpus cavernosum and the retina providing for the drugs therapeutic ben-
efits via the maintenance of the signal transduction signal that leads to a spe-
cific alteration in the phosphorylation status of the cells proteome.
Proteins are modified posttranslationally for a number of different pur-
poses, and protein phosphorylation represents one such modification pro-
cess. Significant other posttranslational modification processes include
glycosylation (the addition of a sugar residue—usually a complex polysac-
charide chain), farnesylation and geranylation (the addition of hydrophobic
Phosphoproteomic Analysis in Biomedicine 29
residues to the protein), and altered redox properties of the target protein
and ubiquitination (usually targeting proteins to the proteasome system
for degradation). The major posttranslation modifications of interest are
phosphorylation, glycosylation, and farnesylation. Ubiquitination represents
a significantly wider and more diverse field of research interest. Of the stated
three posttranslational modifications, a survey of research literature between
2007 and 2012 clearly demonstrates that phosphorylation has been more sig-
nificantly researched compared to the other two modification processes.
Protein phosphorylation articles represent over 70% of the published liter-
ature with glycosylation represented in 16% of the literature and
farnesylation, ubiquitination, and acetylation in 14% (Fig. 2.1). This focus
upon protein phosphorylation analysis can be attributed to a number of dis-
tinct reasons. First, as mentioned previously, the impact of protein phos-
phorylation is prominent in all cells and the mechanism behind the
phosphorylation events is reasonably well understood and appreciated; this
makes the protein phosphorylation event an attractive target therefore for
the study of the development of clinical disorders and also as a therapeutic
target for the treatment of any such disorder. Second, as a posttranslation
modification, protein phosphorylation is a comparatively simple and consis-
tent modification. In mammalian systems, only three amino acids represent
targets of protein phosphorylation (as discussed previously) and the addition
of a single phosphate to any given amino acid is the sole modification.
Phosphorylation
Glycosylation
Farnesylation
Ubiquitination
Acetylation
140
120
100
No. publications
80
60
40
20
0
2007 2008 2009 2010 2011 2012
Year
Figure 2.2 The number of publications related to phosphoproteomics between 2007
and 2012.
Phosphoproteomic Analysis in Biomedicine 31
Brenton, Smith, & Dudley, 2004) and has been utilized to study the function
of newly identified secondary messengers (Bond et al., 2007).
The aim of this review is to first discuss the methodologies available in
order to detect phosphorylated proteins and investigate the site of protein
phosphorylation. Methods that have been developed for the purification
or enrichment of phosphorylated peptides/proteins from proteomes will
also be reviewed as these can be coupled to “traditional” MS proteomic
analysis platforms to provide phosphoproteomic data sets. Following the dis-
cussion of techniques available for phosphoproteomic analyses, examples of
applications in a number of fields will be reviewed in order to provide the
reader with an understanding of the potential of the field to inform biomed-
ical and pharmacological studies in the future.
2. PHOSPHOPROTEOMIC METHODOLOGIES
Traditional methods for studying protein phosphorylations were labo-
rious and time consuming and included techniques such as radiolabeling
of phosphorus atoms, phosphospecific antibodies, and in vitro kinase assays.
These techniques generally involved specific knowledge of the phosphory-
lation sites and phosphate groups. With the advent of MS and supporting
technology, a more rapid and unbiased analysis was developed, allowing
large-scale identification of phosphorylation sites from different model sys-
tems. MS phosphoprotein analysis provides both qualitative and quantitative
analyses to identify and profile the abundance of thousands of phosphopro-
teins in a single experiment. With improvements in separation affinity media
and MS selectivity and sensitivity, there has been an increase in the number
of phosphorylation sites identified (Kanshin, Michnick, & Thibault, 2012)
using specific phosphoproteome databases (Gruhler et al., 2005; Ham
et al., 2008; Mann et al., 2002; Nagaraj, D’Souza, Cox, Olsen, & Mann,
2010; Swaney, McAlister, & Coon, 2008; Syka, Coon, Schroeder,
Shabanowitz, & Hunt, 2004). Protein phosphorylation is a highly dynamic
modification regulated by enzymic control of kinases and phosphatases.
Both intrinsic and extrinsic processes can stimulate proteins to be phosphor-
ylated or dephosphorylated instantly; therefore, sample preparation for
phosphoprotein analyses is an important consideration when planning
method strategies. Inhibitors of both phosphatases and proteases must be
used, and appropriate protein isolation and extraction protocols must be
designed to maintain the integrity, concentration, and phosphorylation sta-
tus of the phosphoproteins from samples. Over the past decade, protocols
Phosphoproteomic Analysis in Biomedicine 33
enriched 100 pmol tryptic a-casein and b-casein digests on both TiO2 and
ZrO2 microtips prior to analysis by negative-ion ESI–FT–ICR (Fourier
transform ion cyclotron resonance) MS. They found more phosphorylated
peptides using ZrO2 and concluded that TiO2 microtips were more selective
for the enrichment of multiply phosphorylated peptides, whereas the ZrO2
tips enriched primarily monophosphorylated peptides. Recently, on-plate
enrichment for MALDI–MS has been developed. Wang and Bruening
(2009) described a method for modification of silicon wafers, which serve as
MALDI plates with 250-mm diameter microspots of phosphopeptide-binding
polymer brushes enclosed by a hydrophobic poly(dimethylsiloxane) layer.
Enrichment resulted in a fivefold decrease in MALDI–MS detection limits
and femtomole-level sensitivity. Zhou, Xu, and Ye (2006) who used zirconium
phosphonate monolayers immobilized on porous silicon observed excellent
selectivity of this approach demonstrated by analyzing phosphopeptides in
the digested mixture of b-casein and BSA with molar ratio of 1:100. Pipette
tip-based off-line TiO2 minicolumns have been widely used for pho-
sphopeptide purification (Bodenmiller et al., 2008; Mohammed et al., 2008;
Ovelleiro, Carrascal, Casas, & Abian, 2009; Wolschin, Wienkoop, &
Weckwerth, 2005). In this regard, Agilent Technologies has introduced a
chip-based device with integrated TiO2 enrichment and RP-LC separation
that is now commercially available (Raijmakers, Kraiczek, de Jong,
Mohammed, & Heck, 2010).
In 1994, Reynolds et al. used an excess of Ca2+ in 50% ethanol for pre-
cipitating phosphopeptides from a tryptic casein hydrolysate (Reynolds,
Riley, & Adamson, 1994). At lower pH, only peptides containing multiple
phosphoserines were enriched. At a pH of 8, all phosphopeptides except two
monophosphorylated ones could be found in the precipitate. Although iron
is used as a central ion in most IMAC methods, other metal ions have been
evaluated for selective phosphate affinity. Posewitz tested different metal
ions including Ga, Sn, Ge, Fe, and others for their applicability in IMAC
phosphopeptide enrichment (Posewitz & Tempst, 1999). With Ga3+, a bet-
ter selectivity compared to conventional Fe3+–IMAC was reported when
analyzing a tryptic digest of phosphoproteins. An interesting approach
was reported from the group of Zhou (Feng et al., 2007; Zhou et al.,
2008). They used a phosphate polymer to coordinatively bind Ti4+ or
Zr4+ ions, and the resulting IMAC resin was used for phosphopeptide
enrichment and compared to Fe3+–IMAC, TiO2, and ZrO2 enrichment
methods. In contrast to MOAC which favors the isolation of mono-
phosphorylated peptides, IMAC was reported to yield a higher proportion
of multiply phosphorylated peptides ( Jensen & Larsen, 2007). The comple-
mentary distribution of phosphopeptides obtainable by TiO2 and Fe(III)–
IMAC can be used advantageously for the combined separation of mono-
phosphorylated and multiply phosphorylated peptides from cell digests.
The sequential use of IMAC and TiO2 also termed SIMAC (sequential elu-
tion from IMAC) gave a twofold increase in phosphopeptide identification
from lysates of human mesenchymal stem cells compared to TiO2 alone
(Thingholm, Jensen, Robinson, & Larsen, 2008). Liang et al. used iTRAQ
(Liang et al., 2007) to compare commercial and prototypal immobilized
metal affinity chelate and metal oxide resins. They tested IMAC magnetic
beads from Invitrogen (Captivate beads), Applied Biosystems (Poros
20 MC beads), and Calbiochem (ProteoExtract) against Nexus tetradentate
metal chelator (Valen Biotech Inc., Atlanta, GA, USA) coupled to
Dynabeads-MyOne (Invitrogen, Carlsbad, CA, USA) tosylactivated beads
(U.S. patent application 20020019496).
charge higher than two. The presence of a phosphate group reduces their
effective charge and their interactions with the SCX resin, resulting in a rel-
ative enrichment of phosphopeptides in early fractions. Beausoleil et al.
(2004) were the first to take advantage of this feature in a phosphoproteomic
study on tryptic digests of HeLa cells where they identified more than 2000
phosphorylation sites using off-line SCX fractionation.
SCX fractionation alone is not sufficient to enrich phosphopeptides from
complex cell extracts, and this technique is typically used to decrease sample
complexity (Imamura, Wakabayashi, & Ishihama, 2012). But, peptides that
have net zero or even negative charge, such as phosphopeptides with basic
residues or multiply phosphorylated peptides, are not well retained on SCX
columns. In order to capture these peptides, ultra acidic SCX exchange was
recently introduced (Hennrich, van den Toorn, Groenewold, Heck, &
Mohammed, 2012), in which tandem SCX is performed under two different
pH conditions (usual and more acidic conditions). Further enrichment of
phosphopeptides from SCX fractions is achieved using IMAC
(Dephoure & Gygi, 2011; Villen, Beausoleil, Gerber, & Gygi, 2007;
Zhai, Villen, Beausoleil, Mintseris, & Gygi, 2008) or MOAC (Olsen
et al., 2006; Sui et al., 2008). The combination of the SCX–IMAC enrich-
ment strategy provided up to 30-fold increase in the proportion of phospho-
peptides observed from Saccharomyces cerevisiae compared to SCX alone
(Villen & Gygi, 2008).
HILIC step to increase selectivity of the setup. Nearly 100% selectivity for
phosphopeptides was achieved when IMAC enrichment was performed
after HILIC chromatography, and over 1000 phosphorylation sites on
914 peptides were identified, demonstrating the use of HILIC as a powerful
prefractionation tool before selective phosphopeptide enrichment is per-
formed. Additionally, phosphopeptides elute at a buffer composition of
70% to 50% ACN containing 0.1% TFA which is asserted to be the optimal
loading buffer composition for IMAC phosphopeptide loading.
The main disadvantage of HILIC is the high organic content of the frac-
tions, which precludes its direct coupling to on-line RP-LC and HILIC is
generally preferred as a prefractionation technique prior to LC–MS analysis
of phosphopeptides. More recent applications of HILIC have been demon-
strated in combination with size-exclusion chromatography to identify low-
abundance phosphoproteins from immunodepleted plasma samples from
prostate cancer patients (Garbis et al., 2011) or with IMAC and stable iso-
tope labeling to profile the abundance of 2857 unique phosphorylation sites
in 1338 phosphoproteins from 1 mg of cell lysates (Wu, Chen, Tai, & Chen,
2011). In contrast, electrostatic repulsion–hydrophilic interaction liquid
chromatography (ERLIC), introduced by Alpert (2008), uses electrostatic
repulsion as an additional chromatographic stationary-phase property to
adjust selectivity in HILIC chromatography. ERLIC makes use of the prop-
erties of HILIC and ion-exchange chromatography whereby the selectivity
is modulated by changing the pH, organic content of mobile phase, or by
applying a salt gradient (Gan, Guo, Zhang, Lim, & Sze, 2008). Anionic
phosphopeptides are preferentially retained on weak anion-exchange col-
umn at pH 2 while neutral and protonated peptides are eluted. At low
pH, carboxyl groups of Glu and Asp and the C-terminus are largely proton-
ated and peptides with positively charged N-termini are electrostatically
repelled from the column. However, negatively charged phosphate groups
of phosphopeptides interact electrostatically with WAX and their retention
times are increased compared with nonphosphopeptides (Chien, Liu, &
Goshe, 2011). In 2011, Chein et al. developed a method utilizing ERLIC,
IMAC, and LC–MS/MS to study Marek’s disease virus (MDV) infection
(Chien et al., 2011). They were able to study the changes occurring in
the phosphoproteome by fractionating peptides from chicken embryo
fibroblast (CEF) digests using ERLIC, then IMAC enrichment to selectively
target phosphorylated peptides prior to LC–MS/MS analysis. Five hundred
and eighty-one unique phosphopeptides were identified from the MDV-
infected CEF samples.
42 Ed Dudley and A. Elizabeth Bond
2.3. MS analysis
After a digested peptide is injected into the MS, a precursor ion is fragmented
into product ions. The abundance and richness of fragmentation ions are
important factors for the effective identification of phosphorylated sites in
shotgun proteomics. Technological development in this area has recently
been very rapid, and very powerful MS instruments have become available.
2.3.2 MALDI–TOF MS
After enrichment, phosphopeptides are subsequently (in the case of MALDI–
TOF MS approach) spotted onto a plate. Selection of matrix crucially
affects phosphopeptide signals. Typically, a-cyano-4-hydroxycinnamic acid
(CHCA) and DHB are used in phosphoproteomics, but the use of other
matrices has also been reported. 2,4,6-Trihydroxyacetophenone (THAP)
with diammonium hydrogen citrate (DAHC) was found to overcome sup-
pression of phosphopeptides by the nonphosphorylated peptides during
positive-ion MALDI–TOF MS analysis compared to CHCA (Yang,
Wu, & Kobayashi, 2004). The abundances of phosphopeptides in tryptic
digests of protein kinase C-treated mouse cardiac troponin I were enhanced
more than 10-fold and using THAP/DAHC leading to the identification of a
unique phosphorylation site. Kjellstr€ om and Jensen (2004) have tested
several organic and inorganic acids as matrix additives to enhance signal
of phosphopeptides in both positive- and negative-ion modes. After exam-
ining phosphoric acid, formic acid, acetic acid, TFA, and heptafluorobutyric
acid, they concluded that 1% phosphoric acid added to DHB significantly
improved the resolution of MALDI mass spectra of intact proteins. According
to Dunn et al. (2006), DHB/phosphoric acid typically results in stronger sig-
nal than CHCA. In our hands, DHB/phosphoric acid also yields stronger
phosphopeptide signals in MS; however, CHCA is more suitable for MS/MS
measurements (A.E. Bond, unpublished data).
Both MALDI and ESI–MS techniques enable the transfer of intact
proteins into the gas phase without fragmentation, but that is all these
two methods have in common. MALDI produces mostly singly charged
44 Ed Dudley and A. Elizabeth Bond
ions and is preferably used with a high mass range analyzer such as the TOF
mass analyzer, while ESI produces multiply charged ions (making larger pro-
teins more accessible to analysis than MALDI does) and can be used with
quadrupoles and ion traps (Fenn, Mann, & Meng, 1989). MALDI is a rapid,
solid-phase technique that can be utilized, for example, in high-throughput
microarrays or imaging of tissue or detection of individual cells or microor-
ganisms. ESI, in contrast, is a liquid technique compatible with on-line
chromatographic techniques and capillary electrophoresis. When coupled
with FT mass spectrometers, it is more sensitive and reaches high perfor-
mance indeed, although the sensitivity of ESI is reduced by the presence
of salts, impurities, and organic buffers, which are more easily tolerated
by MALDI.
3. APPLICATIONS OF PHOSPHOPROTEOMICS
IN BIOMEDICINE
3.1. Applications in cancer research
Phosphoproteomic analyses have been often utilized in the study of dys-
regulation of proliferative pathways which lead to the onset and progression
of various cancers due to the major role that protein phosphorylation usually
plays in the overall control of cellular processes. Phosphoprotein analysis has
commonly been used in order to attempt to obtain a global phosphoprotein
data set with a view to then determining key proteins rather than selectively
46 Ed Dudley and A. Elizabeth Bond
reduce proliferation rates within cancer cells (Yu, Issaq, & Veenstra, 2007).
Bhola et al. (2011) studied the phosphoproteome in head and neck cancer
models which were undergoing proliferation. The proliferative rate was
activated by using gene silencing technology to repress the expression of
the epidermal growth factor receptor (whose express is associated with
reduced tumor growth) while G-protein coupled receptor activation was
ensured by adding an agonist for this receptor type (as these are over-
expressed in such tumors). The study of the phosphoproteomic changes that
were identified as a result of such intervention allowed for the selection of
the protein p70S6K as being more phosphorylated in tumors in which pro-
liferation was activated in this manner (increased sixfold). Therefore, it was
concluded in the study that this protein represents a potential downstream
target of the reduced growth factor receptor/elevated G-protein coupled
receptor activation of cellular proliferation processes. A recent study con-
sidered castration-resistant metastatic prostate cancers taken via biopsy com-
pared to primary prostate tumors which were biopsied prior to any therapy
(Drake et al., 2013). The study profiled phosphotyrosine peptides from the
different samples and highlighted a number of phosphoproteins which cor-
related well with the resistance within tumors which were more aggressive,
including ALK, and MAPK1/3 with a view to developing inhibitors selec-
tive for these particular kinases in such cancers. Further studies have consid-
ered myeloma cancers as the therapeutic target and studied 25 different cell
strains via phosphoproteomic approaches in order to identify tyrosine kinase
receptors specifically phosphorylated (and therefore activated) in such pro-
liferative cell lines as novel targets (Tworkoski et al., 2011). Ovarian cancers
have been studied via similar approaches, with 69 primary cancer cell cul-
tures being utilized and compared (Ren et al., 2012). The study identified
overphosphorylation of the protein ALK in 2–4% of cases and therefore
suggested this protein phosphorylation as a novel target in a number of ovar-
ian cancer cases.
As well as new therapeutic targets, phosphoproteomic analysis has been
applied to investigate resistance mechanisms (as discussed previously) and
identify targets that would reduce resistance in tumors, thereby enhancing
the efficacy of the existing treatment options available to clinicians. An
example of this comes from the field of gastrointestinal cancers and their
treatment with the drug, imatinib. Takahashi et al. (2013) analyzed the phos-
phoproteome of cancers after treatment in order to identify focal adhesion
kinase and associated protein phosphorylation as a key event in the reduction
of the efficacy of the drug on the tumor development (confirmed using
52 Ed Dudley and A. Elizabeth Bond
phosphorylated while the vascular isoform did not present any phosphory-
lation sites being actively modified, suggesting a different function of this
protein within the two different cell types based upon their eventual func-
tion (Namiki, Suzuki, Masuda, Ishihama, & Okano, 2012). Fibroblast
growth factor 2 (FGF-2) acts as a growth factor in human embryonic stem
cells in order to allow for their efficient and effective expansion and produc-
tion. The addition of FGF-2 therefore allows for the stimulation of the stem
cells, and this process was monitored by studying phosphoprotein changes in
the cells, after which 40% of the detected phosphoproteome demonstrated a
significant change in their modification status. The identified proteins
included proteins involved in the self-renewal processes underlying cell pro-
liferation processes and proteins whose expression is regulated by transcrip-
tion factors previously implicated in pluripotency (Zoumaro-Djayoon et al.,
2011). Finally, the role of the phosphoproteome in the clinical disorder,
hemoglobin E/b thalassemia, in which increased apoptosis of erythrocytes
leads to the symptoms of the disorder was undertaken by comparing stem
cells that differentiate to provide these cells on a continual basis in patients
with the disorder compared to control hematopoietic stem cells (Ponnikorn
et al., 2011). The research found that 229 phosphoproteins were found to be
differently presented in the normal and ineffective stem cell lines of which
many were of importance in such cells including cytochrome c and caspase
6—suggesting its direct role in the observed apoptosis symptoms. Within the
discipline of regenerative medicine and further understanding of stem cell
differentiation and production, phosphoproteomics is therefore beginning
to show its value as a targeted method which can be utilized to obtain infor-
mation regarding the function of this posttranslational modification on a
global scale. Further work in this field is envisaged to allow for stem cells
to be more efficiently produced in the laboratory setting and also to provide
a more refined methodology for the control of the differentiation process (in
relation to controlling when this process is initiated and also the nature of the
resulting differentiated cell).
4. DISCUSSION
Following on from advances in the application of modern proteomic
techniques to categorize proteomes and the various changes in expression
60 Ed Dudley and A. Elizabeth Bond
REFERENCES
Aguiar, M., Haas, W., Beausoleil, S. A., Rush, J., & Gygi, S. P. (2010). Gas-phase
rearrangements do not affect site localization reliability in phosphoproteomics data sets.
Journal of Proteome Research, 9, 3103–3107.
Alcolea, M. P., Casado, P., Rodrı́guez-Prados, J. C., Vanhaesebroeck, B., & Cutillas, P. R.
(2012). Phosphoproteomic analysis of leukemia cells under basal and drug-treated con-
ditions identifies markers of kinase pathway activation and mechanisms of resistance.
Molecular & Cellular Proteomics, 11(8), 453–466.
Ali, N. A., & Molloy, M. P. (2011). Quantitative phosphoproteomics of transforming growth
factor-b signaling in colon cancer cells. Proteomics, 11(16), 3390–3401.
Alpert, A. J. (2008). Electrostatic repulsion hydrophilic interaction chromatography for
isocratic separation of charged solutes and selective isolation of phosphopeptides. Ana-
lytical Chemistry, 80, 62–76.
Andersson, L., & Porath, J. (1986). Isolation of phosphoproteins by immobilized metal
(Fe3 +) affinity chromatography. Analytical Biochemistry, 154, 250–254.
Arrigoni, G., Resjo, S., Levander, F., Nilsson, R., Degerman, E., Quadroni, M., et al.
(2006). Chemical derivatization of phosphoserine and phosphothreonine containing
peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS
analysis. Proteomics, 6, 757–766.
Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villén, J., Li, J., et al. (2004).
Large-scale characterization of HeLa cell nuclear phosphoproteins. Proceedings of the National
Academy of Sciences of the United States of America, 101, 12130–12135.
Bhola, N. E., Thomas, S. M., Freilino, M., Joyce, S., Sahu, A., Maxwell, J., et al. (2011).
Targeting GPCR-mediated p70S6K activity may improve head and neck cancer
response to cetuximab. Clinical Cancer Research, 17(15), 4996–5004.
Phosphoproteomic Analysis in Biomedicine 61
Bodenmiller, B., Campbell, D., Gerrits, B., Lam, H., Jovanovic, M., & Picotti, P. (2008).
PhosphoPep—A database of protein phosphorylation sites in model organisms. Nature
Biotechnology, 26, 1339–1340.
Boersema, P. J., Mohammed, S., & Heck, A. J. (2009). Phosphopeptide fragmentation and
analysis by mass spectrometry. Journal of Mass Spectrometry, 44, 861–878.
Bond, A. E., Dudley, E., Tuytten, R., Lemière, F., Smith, C. J., Esmans, E. L., et al. (2007).
Mass spectrometric identification of Rab23 phosphorylation as a response to challenge by
cytidine 3’,5’-cyclic monophosphate in mouse brain. Rapid Communications in Mass Spec-
trometry, 21(16), 2685–2692.
Bond, A. E., Row, P. E., & Dudley, E. (2011). Post-translation modification of proteins;
methodologies and applications in plant sciences. Phytochemistry, 72(10), 975–996.
Brill, L. M., Xiong, W., Lee, K. B., Ficarro, S. B., Crain, A., Xu, Y., et al. (2009). Phos-
phoproteomic analysis of human embryonic stem cells. Cell Stem Cell, 5(2), 204–213.
Casado, P., Alcolea, M. P., Iorio, F., Rodrı́guez-Prados, J. C., Vanhaesebroeck, B., Saez-
Rodriguez, J., et al. (2013). Phosphoproteomics data classify hematological cancer cell
lines according to tumor type and sensitivity to kinase inhibitors. Genome Biology,
14(4), R37.
Chang, Y. W., Chang, Y. T., Wang, Q., Lin, J. J., Chen, Y. J., & Chen, C. C. (2013). Quan-
titative phosphoproteomic study of pressure-overloaded mouse heart reveals dynamin-
related protein 1 as a modulator of cardiac hypertrophy. Molecular & Cellular Proteomics,
12(11), 3094–3107.
Chen, L., Fang, B., Giorgianni, F., Gingrich, J. R., & Beranova-Giorgianni, S. (2011). Inves-
tigation of phosphoprotein signatures of archived prostate cancer tissue specimens via
proteomic analysis. Electrophoresis, 32(15), 1984–1991.
Chien, K. Y., Liu, H. C., & Goshe, M. B. (2011). Development and application of a phos-
phoproteomic method using electrostatic repulsion-hydrophilic interaction chromatog-
raphy (ERLIC), IMAC, and LC-MS/MS analysis to study Marek’s disease virus
infection. Journal of Proteome Research, 10, 4041–4053.
Collins, M. O., Yu, L., Coba, M. P., Husi, H., Campuzano, L., Blackstock, W. P., et al.
(2005). Robust enrichment of phosphorylated species in complex mixtures by sequential
protein and peptide metal-affinity chromatography and analysis by tandem mass spec-
trometry. The Journal of Biological Chemistry, 280, 5972–5982.
Connor, P. A., & McQuillan, A. J. (1999). Phosphate adsorption onto TiO2 from aqueous
solutions: An in situ internal reflection infrared spectroscopic study. Langmuir, 15,
2916–2921.
Dai, J., Wang, L. S., Wu, Y. B., Sheng, Q. H., Wu, J. R., & Shieh, C. H. (2009). Fully
automatic separation and identification of phosphopeptides by continuous
pH-gradient anion exchange online coupled with reversed-phase liquid chromatography
mass spectrometry. Journal of Proteome Research, 8, 133–141.
Deng, N., Zhang, J., Zong, C., Wang, Y., Lu, H., Yang, P., et al. (2011). Phosphoproteome
analysis reveals regulatory sites in major pathways of cardiac mitochondria. Molecular &
Cellular Proteomics, 10(2), M110.000117.
Dephoure, N., & Gygi, S. P. (2011). A solid phase extraction-based platform for rapid phos-
phoproteomic analysis. Methods, 54, 379–386.
Drake, J. M., Graham, N. A., Lee, J. K., Stoyanova, T., Faltermeier, C. M., Sud, S., et al.
(2013). Metastatic castration-resistant prostate cancer reveals intrapatient similarity and
interpatient heterogeneity of therapeutic kinase targets. Proceedings of the National Academy
of Sciences of the United States of America, 110, E4762–E4769.
Dubois, E., Richard, V., Mulder, P., Lamblin, N., Drobecq, H., Henry, J. P., et al. (2011).
Decreased serine207 phosphorylation of troponin T as a biomarker for left ventricular
remodelling after myocardial infarction. European Heart Journal, 32(1), 115–123.
62 Ed Dudley and A. Elizabeth Bond
Dubois-Deruy, E., Belliard, A., Mulder, P., Chwastyniak, M., Beseme, O., Henry, J. P.,
et al. (2013). Circulating plasma serine208-phosphorylated troponin T levels are indica-
tor of cardiac dysfunction. Journal of Cellular and Molecular Medicine, 17, 1335–1344.
http://dx.doi.org/10.1111/jcmm.12112.
Dunn, J. D., Watson, J. T., & Bruening, M. L. (2006). Techniques for phosphopeptide
enrichment prior to analysis by mass spectrometry. Analytical Chemistry, 78, 1574–1580.
Edwards, A. V., Cordwell, S. J., & White, M. Y. (2011). Phosphoproteomic profiling of the
myocyte. Circulation. Cardiovascular Genetics, 4(5), 575.
Feng, H., Ye, M., Zhou, H., Jiang, X., Zou, H., & Gong, B. (2007). Immobilized zirconium
ion affinity chromatography for specific enrichment of phosphopeptides in phospho-
proteome analysis. Molecular Cellular Proteomics, 6, 1656–1665.
Feng, J., Zhu, M., Schaub, M. C., Gehrig, P., Roschitzki, B., Lucchinetti, E., et al. (2008).
Phosphoproteome analysis of isoflurane-protected heart mitochondria: Phosphorylation
of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function. Car-
diovascular Research, 80(1), 20–29.
Fenn, J. B., Mann, M., & Meng, C. K. (1989). Electrospray ionization for mass spectrometry
of large biomolecules. Science, 246, 64–71.
Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., &
Shabanowitz, J. (2002). Phosphoproteome analysis by mass spectrometry and its appli-
cation to Saccharomyces cerevisiae. Nature Biotechnology, 20, 301–305.
Ficarro, S. B., Parikh, J. R., Blank, N. C., & Marto, J. A. (2008). Niobium(V) oxide
(Nb2O5): Application to phosphoproteomics. Analytical Chemistry, 80, 4606–4613.
Ficarro, S. B., Zhang, Y., Carrasco-Alfonso, M. J., Garg, B., Adelmant, G., & Webber, J. T.
(2011). Online nanoflow multidimensional fractionation for high efficiency pho-
sphopeptide analysis. Molecular and Cellular Proteomics, 10, O111.011064.
Frackelton, A. R., Jr., Ross, A. H., & Eisen, H. N. (1983). Characterization and use of mono-
clonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed
cells and growth factor-stimulated cells. Molecular and Cellular Biology, 3, 1343–1352.
Frederick, M. J., VanMeter, A. J., Gadhikar, M. A., Henderson, Y. C., Yao, H.,
Pickering, C. C., et al. (2011). Phosphoproteomic analysis of signaling pathways in head
and neck squamous cell carcinoma patient samples. The American Journal of Pathology,
178(2), 548–571.
Gan, C. S., Guo, T., Zhang, H., Lim, S. K., & Sze, S. K. (2008). A comparative study of
electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) versus SCX-
IMAC-based methods for phosphopeptide isolation/enrichment. Journal of Proteome
Research, 7, 4869–4877.
Garbis, S. D., Roumeliotis, T. I., Tyritzis, S. I., Zorpas, K. M., Pavlakis, K., &
Constantinides, C. A. (2011). A novel multidimensional protein identification technol-
ogy approach combining protein size exclusion prefractionation, peptide zwitterion-ion
hydrophilic interaction chromatography, and nano-ultraperformance RP chromatogra-
phy/nESI-MS2 for the in-depth analysis of the serum proteome and phosphoproteome:
Application to clinical sera derived from humans with benign prostate hyperplasia. Ana-
lytical Chemistry, 83, 708–718.
Gratia, S., Kay, L., Michelland, S., Sève, M., Schlattner, U., & Tokarska-Schlattner, M.
(2012). Cardiac phosphoproteome reveals cell signaling events involved in doxorubicin
cardiotoxicity. Journal of Proteomics, 75(15), 4705–4716.
Grimes, M. L., Lee, W. J., van der Maaten, L., & Shannon, P. (2013). Wrangling phospho-
proteomic data to elucidate cancer signaling pathways. PLoS One, 8(1), e52884.
Gruhler, A., Olsen, J. V., Mohammed, S., Mortensen, P., Faergeman, N. J., Mann, M., et al.
(2005). Quantitative phosphoproteomics applied to the yeast pheromone signalling
pathway. Molecular and Cellular Proteomics, 4, 310–327.
Phosphoproteomic Analysis in Biomedicine 63
Guimond, D. M., Cam, N. R., Hirve, N., Duan, W., Lambris, J. D., Croft, M., et al. (2013).
Regulation of immune responsiveness in vivo by disrupting an early T-cell signaling
event using a cell-permeable peptide. PLoS One, 8(5), e63645.
Guo, H., Isserlin, R., Chen, X., Wang, W., Phanse, S., Zandstra, P. W., et al. (2013). Inte-
grative network analysis of signaling in human CD34(+) hematopoietic progenitor cells
by global phosphoproteomic profiling using TiO2 enrichment combined with 2D
LC-MS/MS and pathway mapping. Proteomics, 13(8), 1325–1333.
Guo, T., Lee, S. S., Ng, W. H., Zhu, Y., Gan, C. S., Zhu, J., et al. (2011). Global molecular
dysfunctions in gastric cancer revealed by an integrated analysis of the phosphoproteome
and transcriptome. Cellular and Molecular Life Sciences, 68(11), 1983–2002.
Ham, B. M., Yang, F., Jayachandran, H., Jaitly, N., Monroe, M. E., & Gritsenko, M. A.
(2008). The influence of sample preparation and replicate analyses on HeLa cell phos-
phoproteome coverage. Journal of Proteome Research, 7, 2215–2221.
Han, G., Ye, M., Zhou, H., Jiang, X., Feng, S., & Jiang, X. (2008). Large-scale phos-
phoproteome analysis of human liver tissue by enrichment and fractionation of
phosphopeptides with strong anion exchange chromatography. Proteomics, 8,
1346–1361.
Hekmat, O., Munk, S., Fogh, L., Yadav, R., Francavilla, C., Horn, H., et al. (2013). TIMP-1
increases expression and phosphorylation of proteins associated with drug resistance in
breast cancer cells. Journal of Proteome Research, 12(9), 4136–4151.
Hennrich, M. L., van den Toorn, H. W., Groenewold, V., Heck, A. J., & Mohammed, S.
(2012). Ultra acidic strong cation exchange enabling the efficient enrichment of basic
phosphopeptides. Analytical Chemistry, 84, 1804–1808.
Hilger, M., Bonaldi, T., Gnad, F., & Mann, M. (2009). Systems-wide analysis of a phospha-
tase knock-down by quantitative proteomics and phosphoproteomics. Molecular and Cel-
lular Proteomics, 8, 1908–1920.
Huang, S. S., Clarke, D. C., Gosline, S. J., Labadorf, A., Chouinard, C. R., Gordon, W.,
et al. (2013). Linking proteomic and transcriptional data through the interactome and
epigenome reveals a map of oncogene-induced signaling. PLoS Computational Biology,
9(2), e1002887.
Hunt, D. F., Buko, A. M., Ballard, J. M., Shabanowitz, J., & Giordani, A. B. (1981).
Sequence analysis of polypeptides by collision activated dissociation on a triple quadru-
pole mass spectrometer. Biomedical Mass Spectrometry, 8, 397–408.
Huttlin, E. L., Jedrychowski, M. P., Elias, J. E., Goswami, T., Rad, R., & Beausoleil, S. A.
(2010). A tissue-specific atlas of mouse protein phosphorylation and expression. Cell,
143, 1174–1189.
Ikeguchi, Y., & Nakamua, H. (1997). Determination of organic phosphates by column-
switching high performance anion-exchange chromatography using on-line
preconcentration on titania. Analytical Sciences, 13, 479–483.
Ikeguchi, Y., & Nakamura, H. (2000). Selective enrichment of phospholipids by titania.
Analytical Sciences, 16, 541–543.
Imamura, H., Wakabayashi, M., & Ishihama, Y. (2012). Analytical strategies for shotgun
phosphoproteomics: Status and prospects. Seminars in Cell & Developmental Biology, 23,
836–842.
Iwai, L. K., Payne, L. S., Luczynski, M. T., Chang, F., Xu, H., Clinton, R. W., et al. (2013).
Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation
downstream of wild-type DDR2 and its lung cancer mutants. The Biochemical Journal,
454(3), 501–513.
Jaffe, H., Veeranna, & Pant, H. C. (1998). Characterization of the phosphorylation sites of
human high molecular weight neurofilament protein by electrospray ionization tandem
mass spectrometry and database searching. Biochemistry, 37, 16211–16224.
64 Ed Dudley and A. Elizabeth Bond
Jedrychowski, M. P., Huttlin, E. L., Haas, W., Sowa, M. E., Rad, R., & Gygi, S. P. (2011).
Evaluation of HCD- and CID-type fragmentation within their respective detection plat-
forms for murine phosphoproteomics. Molecular and Cellular Proteomics, 10, 1–19.
Jensen, S. S., & Larsen, M. R. (2007). Evaluation of the impact of some experimental pro-
cedures on different phosphopeptide enrichment techniques. Rapid Communications in
Mass Spectrometry, 21, 3635–3645.
Jindra, P. T., Hsueh, A., Hong, L., Gjertson, D., Shen, X. D., Gao, F., et al. (2008). Anti-
MHC class I antibody activation of proliferation and survival signaling in murine cardiac
allografts. Journal of Immunology, 180(4), 2214–2224.
Kanshin, E., Michnick, S., & Thibault, P. (2012). Sample preparation and analytical strategies
for large-scale phosphoproteomics experiments. Seminars in Cell & Developmental Biology,
23, 843–853.
Kawahara, M., Nakamura, H., & Nakajima, T. (1989). Group separation of ribonucleosides
and deoxyribonucleosides on a new ceramic titania column. Analytical Sciences, 5,
485–486.
Kim, J., Kim, J. S., Kim, H. E., Jeon, Y. J., Kim, D. W., Soh, Y., et al. (2011). Proteomic
analysis of phosphotyrosyl proteins in human embryonic stem cell-derived neural stem
cells. Neuroscience Letters, 499(3), 158–163.
Kjellstr€
om, S., & Jensen, O. N. (2004). Phosphoric acid as a matrix additive for MALDI MS
analysis of phosphopeptides and phosphoproteins. Analytical Chemistry, 76, 5109–5117.
Klammer, M., Kaminski, M., Zedler, A., Oppermann, F., Blencke, S., Marx, S., et al. (2012).
Phosphosignature predicts dasatinib response in non-small cell lung cancer. Molecular &
Cellular Proteomics, 11(9), 651–668.
Knight, Z. A., Schilling, B., Row, R. H., Kenski, D. M., Gibson, B. W., & Shokat, K. M.
(2003). Phosphospecific proteolysis for mapping sites of protein phosphorylation. Nature
Biotechnology, 21, 1047–1054.
Kokubu, M., Ishihama, Y., Sato, T., Nagasu, T., & Oda, Y. (2005). Specificity of
immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for
protein phosphorylation analysis. Analytical Chemistry, 77, 5144–5154.
Kooij, V., Holewinski, R. J., Murphy, A. M., & Van Eyk, J. E. (2013). Characterization of
the cardiac myosin binding protein-C phosphoproteome in healthy and failing human
hearts. Journal of Molecular and Cellular Cardiology, 60, 116–120.
Kwei, K. A., Baker, J. B., & Pelham, R. J. (2012). Modulators of sensitivity and resistance to
inhibition of PI3K identified in a pharmacogenomic screen of the NCI-60 human tumor
cell line collection. PLoS One, 7(9), e46518.
Kweon, H. K., & Håkansson, K. (2006). Selective zirconium dioxide-based enrichment of
phosphorylated peptides for mass spectrometric analysis. Analytical Chemistry, 78,
1743–1749.
Kyono, Y., Sugiyama, N., Imami, K., Tomita, M., & Ishihama, Y. (2008). Successive and
selective release of phosphorylated peptides captured by hydroxy acid-modified metal
oxide chromatography. Journal of Proteome Research, 7, 4585–4593.
Larsen, M. R., Thingholm, T. E., Jensen, O. N., Roepstorff, P., & Jorgensen, T. J. (2005).
Highly selective enrichment of phosphorylated peptides from peptide mixtures using
titanium dioxide microcolumns. Molecular and Cellular Proteomics, 4, 873–886.
Laurence, A., Astoul, E., Hanrahan, S., Totty, N., & Cantrell, D. (2004). Identification of
pro-interleukin 16 as a novel target of MAP kinases in activated T lymphocytes. European
Journal of Immunology, 34(2), 587–597.
Lee, B. Y., Hochgräfe, F., Lin, H. M., Castillo, L., Wu, J., Raftery, M. J., et al. (2014). Phos-
phoproteomic profiling identifies focal adhesion kinase as a mediator of docetaxel resis-
tance in castrate resistant prostate cancer. Molecular Cancer Therapeutics, 13, 190–201.
Leitner, A., & Leitner, W. (2009). Chemical tagging strategies for mass spectrometry-based
phosphoproteomics. Methods in Molecular Biology, 527, 229–243.
Phosphoproteomic Analysis in Biomedicine 65
Leitner, A., Sturm, M., Smått, J. H., Järn, M., & Lindén, M. (2009). Optimizing the perfor-
mance of tin dioxide microspheres for phosphopeptide enrichment. Analytica Chimica
Acta, 6(38), 51–57.
Li, X., Gerber, S. A., Rudner, A. D., Beausoleil, S. A., Haas, W., Villén, J., et al. (2007).
Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae.
Journal of Proteome Research, 6, 1190–1197.
Liang, X. Q., Fonnum, G., Hajivandi, M., Stene, T., Kjus, N. H., & Ragnhildstveit, E.
(2007). Quantitative comparison of IMAC and TiO2 surfaces used in the study of reg-
ulated, dynamic protein phosphorylation. Journal of the American Society for Mass Spectrom-
etry, 18, 1932–1944.
Lo, T., Tsai, C. F., Shih, Y. R., Wang, Y. T., Lu, S. C., Sung, T. Y., et al. (2012). Phos-
phoproteomic analysis of human mesenchymal stromal cells during osteogenic differen-
tiation. Journal of Proteome Research, 11(2), 586–598.
Mann, M., Ong, S. E., Gronborg, M., Steen, H., Jensen, O. N., & Pandey, A. (2002). Anal-
ysis of protein phosphorylation using mass spectrometry: Deciphering the phospho-
proteome. Trends in Biotechnology, 20, 261–268.
Matsuda, H., Nakamura, H., & Nakajima, T. (1990). New ceramic titania selective adsorbent
for organic phosphates. Analytical Sciences, 6, 911–912.
Mazanek, M., Mitulovic, G., Herzog, F., Stingl, C., Hutchins, J. R. A., Peters, J.-M., et al.
(2007). Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chro-
matography prior to HPLC-MS/MS analysis. Nature Protocols, 2, 1059–1069.
Mazanek, M., Roitinger, E., Hudecz, O., Hutchins, J. R. A., Hegemann, B., Mit-ulovic, G.,
et al. (2010). A new acid mix enhances phosphopeptide enrichment on titanium- and
zirconium dioxide for mapping of phosphorylation sites on protein complexes. Journal
of Chromatography B, 878, 515–524.
McDonnell, S. R., Hwang, S. R., Rolland, D., Murga-Zamalloa, C., Basrur, V.,
Conlon, K. P., et al. (2013). Integrated phosphoproteomic and metabolomic profiling
reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming
in anaplastic large cell lymphoma. Blood, 122(6), 958–968.
McMurray, J. S., Mandal, P. K., Liao, W. S., Klostergaard, J., & Robertson, F. M. (2012).
The consequences of selective inhibition of signal transducer and activator of transcrip-
tion 3 (STAT3) tyrosine705 phosphorylation by phosphopeptide mimetic prodrugs
targeting the Src homology 2 (SH2) domain. JAKSTAT, 1(4), 263–347.
McNulty, D. E., & Annan, R. S. (2008). Hydrophilic interaction chromatography reduces
the complexity of the phosphoproteome and improves global phosphopeptide isolation
and detection. Molecular and Cellular Proteomics, 7, 971–980.
Mohammed, S., Kraiczek, K., Pinkse, M. W. H., Lemeer, S., Benschop, J. J., &
Heck, A. J. R. (2008). Chip-based enrichment and nanoLC-MS/MS analysis of phos-
phopeptides from whole lysates. Journal of Proteome Research, 7, 1565–1571.
Myung, J. K., & Sadar, M. D. (2012). Large scale phosphoproteome analysis of LNCaP
human prostate cancer cells. Molecular Biosystems, 8(8), 2174–2182.
Nagaraj, N., D’Souza, R. C., Cox, J., Olsen, J. V., & Mann, M. (2010). Feasibility of large-
scale phosphoproteomics with higher energy collisional dissociation fragmentation. Jour-
nal of Proteome Research, 9, 6786–6794.
Namiki, J., Suzuki, S., Masuda, T., Ishihama, Y., & Okano, H. (2012). Nestin protein is
phosphorylated in adult neural stem/progenitor cells and not endothelial progenitor
cells. Stem Cells International, 2012, 430138.
Newton, R. P., Brenton, A. G., Smith, C. J., & Dudley, E. (2004). Plant proteome analysis
by mass spectrometry: Principles, problems, pitfalls and recent developments.
Phytochemistry, 65(11), 1449–1485.
Nicolaou, P., Rodriguez, P., Ren, X., Zhou, X., Qian, J., Sadayappan, S., et al. (2009).
Inducible expression of active protein phosphatase-1 inhibitor-1 enhances basal cardiac
66 Ed Dudley and A. Elizabeth Bond
Reynolds, E. C., Riley, P. F., & Adamson, N. J. (1994). A selective precipitation purification
procedure for multiple phosphoseryl-containing peptides and methods for their identi-
fication. Analytical Biochemistry, 217, 277–284.
Rigbolt, K. T., Prokhorova, T. A., Akimov, V., Henningsen, J., Johansen, P. T., Kratchmarova, I.,
et al. (2011). System-wide temporal characterization of the proteome and phosphoproteome
of human embryonic stem cell differentiation. Science Signaling, 4(164), rs3.
Rivera, J. G., Choi, Y. S., Vujcic, S., Wood, T. D., & Colón, L. A. (2009). Enrichment/
isolation of phosphorylated peptides on hafnium oxide prior to mass spectrometric anal-
ysis. Analyst, 134, 31–33.
Ruperez, P., Gago-Martinez, A., Burlingame, A. L., & Oses-Prieto, J. A. (2012). Quanti-
tative phosphoproteomic analysis reveals a role for serine and threonine kinases in the
cytoskeletal reorganization in early T cell receptor activation in human primary
T cells. Molecular & Cellular Proteomics, 11(5), 171–186.
Rush, J., Moritz, A., Lee, K. A., Guo, A., Goss, V. L., Spek, E. J., et al. (2005).
Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nature Biotechnol-
ogy, 23, 94–101.
Salih, E. (2005). Phosphoproteomics by mass spectrometry and classical protein chemistry
approaches. Mass Spectrometry Reviews, 24, 828–846.
Savitski, M. M., Lemeer, S., & Boesche, M. (2011). Confident phosphorylation site locali-
zation using the Mascot Delta Score. Molecular & Cellular Proteomics, 10, M110.003830.
Schreiber, T. B., Mausbacher, N., Soroka, J., Wandinger, S. K., Buchner, J., & Daub, H.
(2012). Global analysis of phosphoproteome regulation by the Ser/Thr phosphatase
Ppt1 in Saccharomyces cerevisiae. Journal of Proteome Research, 11, 2397–2408.
Schroeder, M. J., Shabanowitz, J., Schwartz, J. C., Hunt, D. F., & Coon, J. J. (2004).
A neutral loss activation method for improved phosphopeptide sequence analysis by
quadrupole ion trap mass spectrometry. Analytical Chemistry, 76, 3590–3598.
Simon, E. S., Young, M., Chan, A., Bao, Z. Q., & Andrews, P. C. (2008). Improved enrich-
ment strategies for phosphorylated peptides on titanium dioxide using methyl esterifica-
tion and pH gradient elution. Analytical Biochemistry, 377, 234–242.
Ståhl, S., Branca, R. M., Efazat, G., Ruzzene, M., Zhivotovsky, B., Lewensohn, R., et al.
(2011). Phosphoproteomic profiling of NSCLC cells reveals that ephrin B3 regulates
pro-survival signaling through Akt1-mediated phosphorylation of the EphA2 receptor.
Journal of Proteome Research, 10(5), 2566–2578.
Steen, H., Kuster, B., & Fernandez, M. (2001). Detection of tyrosine phosphorylated pep-
tides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion
mode. Analytical Chemistry, 73, 1440–1448.
Sugiyama, N., Masuda, T., Shinoda, K., Nakamura, A., Tomita, M., & Ishihama, Y. (2007).
Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chroma-
tography for nano-LC–MS/MS in proteomics applications. Molecular and Cellular Prote-
omics, 6, 1103–1109.
Sui, S., Wang, J., Yang, B., Song, L., Zhang, J., & Chen, M. (2008). Phosphoproteome anal-
ysis of the human Chang liver cells using SCX and a complementary mass spectrometric
strategy. Proteomics, 8, 2024–2034.
Swaney, D. L., McAlister, G. C., & Coon, J. J. (2008). Decision tree-driven tandem mass
spectrometry for shotgun proteomics. Nature Methods, 5, 959–964.
Syka, J. E., Coon, J. J., Schroeder, M. J., Shabanowitz, J., & Hunt, D. F. (2004). Peptide and
protein sequence analysis by electron transfer dissociation mass spectrometry. Proceedings
of the National Academy of Sciences of the United States of America, 101, 9528–9533.
Takahashi, T., Serada, S., Ako, M., Fujimoto, M., Miyazaki, Y., Nakatsuka, R., et al. (2013).
New findings of kinase switching in gastrointestinal stromal tumor under imatinib using
phosphoproteomic analysis. International Journal of Cancer, 133(11), 2737–2743.
68 Ed Dudley and A. Elizabeth Bond
Takano, S., Sogawa, K., Yoshitomi, H., Shida, T., Mogushi, K., Kimura, F., et al. (2010).
Increased circulating cell signalling phosphoproteins in sera are useful for the detection of
pancreatic cancer. British Journal of Cancer, 103(2), 223–231.
Tani, K., & Suzuki, Y. (1997). Investigation of the ion-exchange behaviour of titania-
application as a packing material for ion chromatography. Chromatographia, 46, 623–627.
Thingholm, T. E., Jensen, O. N., Robinson, P. J., & Larsen, M. R. (2008). SIMAC (sequen-
tial elution from IMAC), a phosphoproteomics strategy for the rapid separation of mono-
phosphorylated from multiply phosphorylated peptides. Molecular and Cellular Proteomics,
7, 661–671.
Thingholm, T. E., Jorgensen, T. J., & Jensen, O. N. (2006). Highly selective enrichment of
phosphorylated peptides using titanium dioxide. Nature Protocols, 1, 1929–1935.
Tobe, B. T., Hou, J., Crain, A. M., Singec, I., Snyder, E. Y., & Brill, L. M. (2012). Phos-
phoproteomic analysis: An emerging role in deciphering cellular signaling in human
embryonic stem cells and their differentiated derivatives. Stem Cell Reviews, 8(1), 16–31.
Tworkoski, K., Singhal, G., Szpakowski, S., Zito, C. I., Bacchiocchi, A., Muthusamy, V.,
et al. (2011). Phosphoproteomic screen identifies potential therapeutic targets in mela-
noma. Molecular Cancer Research, 9(6), 801–812.
Vazquez-Martin, A., Oliveras-Ferraros, C., Colomer, R., Brunet, J., & Menendez, J. A.
(2008). Low-scale phosphoproteome analyses identify the mTOR effector p70 S6 kinase
1 as a specific biomarker of the dual-HER1/HER2 tyrosine kinase inhibitor lapatinib
(Tykerb) in human breast carcinoma cells. Annals of Oncology, 19(6), 1097–1109.
Villen, J., Beausoleil, S. A., Gerber, S. A., & Gygi, S. P. (2007). Large-scale phosphorylation
analysis of mouse liver. Proceedings of the National Academy of Sciences of the United States of
America, 104, 1488–1493.
Villen, J., & Gygi, S. P. (2008). The SCX/IMAC enrichment approach for global phosphor-
ylation analysis by mass spectrometry. Nature Protocols, 3, 1630–1638.
Wang, W. H., & Bruening, M. L. (2009). Phosphopeptide enrichment on functionalized
polymer microspots for MALDI-MS analysis. Analyst, 134, 512–518.
Wijeratne, A. B., Manning, J. R., Schultz Jel, J., & Greis, K. D. (2013). Quantitative phos-
phoproteomics using acetone-based peptide labeling: Method evaluation and application
to a cardiac ischemia/reperfusion mode. Journal of Proteome Research, 12, 4268–4279.
Winck, F. V., Belloni, M., Pauletti, B. A., de Lima Zanella, J., Domingues, R. R.,
Sherman, N. E., et al. (2014). Phosphoproteome analysis reveals differences in phos-
phosite profiles between tumorigenic and non-tumorigenic epithelial cells. Journal of
Proteomics, 96, 67–81 S1874-3919(13)00554-X.
Wolschin, F., Wienkoop, S., & Weckwerth, W. (2005). Enrichment of phosphorylated pro-
teins and peptides from complex mixtures using metal oxide/hydroxide affinity chroma-
tography (MOAC). Proteomics, 5, 4389–4397.
Wu, C. J., Chen, Y. W., Tai, J. H., & Chen, S. H. (2011). Quantitative phosphoproteomics
studies using stable A isotope dimethyl labelling coupled with IMAC-HILIC-nanoLC–
MS/MS for estrogen-induced transcriptional regulation. Journal of Proteome Research, 10,
1088–1097.
Wu, H. Y., Tseng, V. S., Chen, L. C., Chang, H. Y., Chuang, I. C., Tsay, Y. G., et al.
(2010). Identification of tyrosine-phosphorylated proteins associated with lung cancer
metastasis using label-free quantitative analyses. Journal of Proteome Research, 9(8),
4102–4112.
Xie, X., Feng, S., Vuong, H., Liu, Y., Goodison, S., & Lubman, D. M. (2010).
A comparative phosphoproteomic analysis of a human tumor metastasis model using a
label-free quantitative approach. Electrophoresis, 31(11), 1842–1852.
Yan, G. R., Ding, W., Xu, S. H., Xu, Z., Xiao, C. L., Yin, X. F., et al. (2011). Character-
ization of phosphoproteins in gastric cancer secretome. OMICS, 15(1–2), 83–90.
Phosphoproteomic Analysis in Biomedicine 69
Yang, X. F., Wu, X. P., & Kobayashi, T. (2004). Enhanced ionization of phosphorylated
peptides during MALDI TOF mass spectrometry. Analytical Chemistry, 76, 1532–1536.
Yu, L. R., Issaq, H. J., & Veenstra, T. D. (2007). Phosphoproteomics for the discovery of
kinases as cancer biomarkers and drug targets. Proteomics Clinical Applications, 1(9),
1042–1057.
Yu, G., Xiao, C. L., Lu, C. H., Jia, H. T., Ge, F., Wang, W., et al. (2011). Phosphoproteome
profile of human lung cancer cell line A549. Molecular Biosystems, 7(2), 472–479.
Zakharchenko, O., Cojoc, M., Dubrovska, A., & Souchelnytskyi, S. (2013). A role of
TGFß1 dependent 14-3-3s phosphorylation at Ser69 and Ser74 in the regulation of
gene transcription, stemness and radioresistance. PLoS One, 8(5), e65163.
Zanivan, S., Meves, A., Behrendt, K., Schoof, E. M., Neilson, L. J., Cox, J., et al. (2013).
In vivo SILAC-based proteomics reveals phosphoproteome changes during mouse skin
carcinogenesis. Cell Reports, 3(2), 552–566.
Zhai, B., Villen, J., Beausoleil, S. A., Mintseris, J., & Gygi, S. P. (2008). Phosphoproteome
analysis of Drosophila melanogaster embryos. Journal of Proteome Research, 7, 1675–1682.
Zhang, K. (2006). From purification of large amounts of phospho-compounds (nucleotides)
to enrichment of phosphopeptides using anion-exchanging resin. Analytical Biochemistry,
357, 225–231.
Zhong, J., Kim, M. S., Chaerkady, R., Wu, X., Huang, T. C., Getnet, D., et al. (2012).
TSLP signaling network revealed by SILAC-based phosphoproteomics. Molecular &
Cellular Proteomics, 11(6), M112.017764.
Zhou, H., Xu, S., & Ye, M. (2006). Zirconium phosphonate-modified porous silicon for
highly specific capture of phosphopeptides and MALDI-TOF MS analysis. Journal of Pro-
teome Research, 5, 2431–2437.
Zhou, H., Ye, M., Dong, J., Han, G., Jiang, X., Wu, R., et al. (2008). Specific pho-
sphopeptide enrichment with immobilized titanium ion affinity chromatography adsor-
bent for phosphoproteome analysis. Journal of Proteome Research, 7, 3957–3967.
Zimman, A., Chen, S. S., Komisopoulou, E., Titz, B., Martı́nez-Pinna, R., Kafi, A., et al.
(2010). Activation of aortic endothelial cells by oxidized phospholipids:
A phosphoproteomic analysis. Journal of Proteome Research, 9(6), 2812–2824.
Zoumaro-Djayoon, A. D., Ding, V., Foong, L. Y., Choo, A., Heck, A. J., & Muñoz, J.
(2011). Investigating the role of FGF-2 in stem cell maintenance by global phospho-
proteomics profiling. Proteomics, 11(20), 3962–3971.
CHAPTER THREE
Contents
1. Introduction 72
2. Glycoproteomic Profiling by MS 75
2.1 Glycoproteomics methodology 75
2.2 Affinity enrichment 76
2.3 Glycoprotein digestion 82
2.4 Glycan release 83
2.5 Chromatographic separation and SPE 85
2.6 Mass spectrometry 88
2.7 Quantitation 93
2.8 Bioinformatics 98
3. MS-Based Glycoproteomics in Disease Research 99
3.1 Cancer biomarker research 99
3.2 Neurodegenerative disease research 104
4. Concluding Remarks 106
Acknowledgments 107
References 107
Abstract
Protein glycosylation plays fundamental roles in many biological processes as one of the
most common, and the most complex, posttranslational modification. Alterations in gly-
cosylation profile are now known to be associated with many diseases. As a result, the
discovery and detailed characterization of glycoprotein disease biomarkers is a primary
interest of biomedical research. Advances in mass spectrometry (MS)-based glyco-
proteomics and glycomics are increasingly enabling qualitative and quantitative
approaches for site-specific structural analysis of protein glycosylation. While the com-
plexity presented by glycan heterogeneity and the wide dynamic range of clinically rel-
evant samples like plasma, serum, cerebrospinal fluid, and tissue make comprehensive
analyses of the glycoproteome a challenging task, the ongoing efforts into the devel-
opment of glycoprotein enrichment, enzymatic digestion, and separation strategies
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 71
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00003-8
72 Dustin C. Frost and Lingjun Li
1. INTRODUCTION
N-linked glycans
Chitobiose Bisecting
core GlcNAc
β6 β6
α6 α6
β3 β3 β3 β3 β3 α3
2. GLYCOPROTEOMIC PROFILING BY MS
2.1. Glycoproteomics methodology
Because the success of any MS experiment relies heavily on analyte purity,
the ultimate aim of sample preparation in an MS-based glycoproteomics
workflow is to simplify or purify a sample to facilitate sensitive detection
of peptides and glycans by the mass spectrometer. Once proteins are
harvested from biological specimens, glycoproteins must be isolated from
nonglycosylated proteins. Top-down MS analysis of purified glycoproteins
can be performed, but bottom-up strategies, in which glycoproteins are
digested into peptides and glycopeptides prior to MS analysis, are most com-
mon. Mixtures of glycopeptides and nonglycosylated peptides present a
problem, however. The hydrophilic nature of attached glycans significantly
impairs the ionization of glycopeptides, and the nonglycosylated peptides are
preferentially ionized and detected by a great degree. The combination of
enrichment and chromatography serves to sufficiently isolate the glycopep-
tides of interest. Glycan cleavage, followed by derivatization and separation,
allows detailed glycomics characterization of composition, structure, link-
ages, and isomers by tandem mass spectrometry (MS/MS), though the
76 Dustin C. Frost and Lingjun Li
Glycoprotein enrichment
Proteolysis
Gylcopeptides
and peptides
Glycopeptide
enrichment/separation
Glycopeptides
Peptides Glycans
Bioinformatics
Glycoprotein identification
Glycan characterization
Glycosylation site assignment
Quantitation
Boronic acid chemistry has been used for glycopeptide enrichment based
on its covalent, yet reversible, chemical reaction with 1-2 and 1-3 cis-diol con-
taining saccharides (e.g., Man, Glc, and Gal) to form stable cyclic esters
(Sparbier et al., 2005; Sparbier, Wenzel, & Kostrzewa, 2006). Binding occurs
under basic or nonaqueous conditions, and elution under acidic conditions
yields the glycopeptides with native glycans still attached. Boronic acid recog-
nition of glycans is nonspecific and tolerant of the various branching and linear
glycans as well as monosaccharide modifications, enabling unbiased enrich-
ment of a wide range of N- and O-linked glycopeptides. The covalent inter-
action with glycosylated peptides allows stringent washing conditions at pH
>8. Boronic acid can be easily functionalized to a variety of supports such
as mesoporous silica (Xu et al., 2009), monoliths (Huang et al., 2013), and
nanoparticles (Pan, Sun, Zheng, & Yang, 2013; Zhang, Xu, et al., 2009;
Zhou et al., 2008) for use with HPLC and capillary columns (Zhang et al.,
2008, 2007), pipette tips (Takátsy et al., 2009), and matrix-assisted laser
desorption–ionization (MALDI) plates (Tang et al., 2009; Xu, Zhang,
Lu, & Yang, 2010).
Titanium dioxide (TiO2) is used in glycopeptide enrichment and solid-
phase extraction (SPE) applications due to its affinity for sialic acid (Larsen
et al., 2007; Palmisano et al., 2010; Zhang, Sheng, et al., 2011). As both
phosphopeptides and glycopeptides bind to TiO2, phosphatase pretreatment
to removed phosphate modifications benefits glycopeptide enrichment effi-
ciency (Larsen et al., 2007). Binding of sialic acid to TiO2 occurs by way of
negative charges on the carboxylic acid and hydroxyl groups of sialic acid
that form a multidentate chelating ligand to Ti4+. The specificity toward
sialic acid is especially attractive in that increased glycan sialylation has been
associated with cancer progression, hepatitis, and inflammation (Larsen
et al., 2007; Mondal, Chatterjee, Chawla, & Chatterjee, 2011; Nie, Li, &
Sun, 2012).
Antibody-based strategies are especially useful when a single glycopro-
tein target needs to be isolated. However, because glycans are poor antigens,
it is difficult to obtain antiglycan antibodies with sufficient affinity and spec-
ificity to use for enrichment purposes. Still, a number of antibodies with rel-
evant antigens in O-GlcNAc (Comer, Vosseller, Wells, Accavitti, & Hart,
2001; Wang, Pandey, & Hart, 2007), O-GalNAc (Nakada et al., 1991), sialyl
LewisX (Cho, Jung, & Regnier, 2008), and polysialic acid (Liedtke et al.,
2001) have been used effectively for glycoprotein enrichment. Recently,
Teo et al. were able to procure three monoclonal antibodies against
O-GlcNAc using a synthetic antigen and enrich three subsets of potentially
82 Dustin C. Frost and Lingjun Li
Proteins that are poorly digested by trypsin alone have been successfully
analyzed following digestion with chymotrypsin (Grass, Pabst, Chang,
Wozny, & Altmann, 2011; Nyalwidhe et al., 2013), pepsin (Taga,
Kusubata, Ogawa-Goto, & Hattori, 2013), and Glu C–trypsin mix
(Pompach, Chandler, Lan, Edwards, & Goldman, 2012). In a complex
glycoproteomics experiment, Chen et al. demonstrated that pepsin and
thermolysin digestion complemented trypsin digestion for human liver tis-
sue samples, increasing the number of identified glycosites by half (Chen
et al., 2009). The nonspecific proteinase K and broadly specific pronase (a
protease cocktail) produce short glycopeptides three to eight amino acids
in length that are perhaps more useful for site-specific glycosylation analysis
(Clowers, Dodds, Seipert, & Lebrilla, 2007; Temporini et al., 2007). The
resulting glycans with short amino acid sequence “tags” are then appropriate
for proved glycan separation techniques like hydrophilic interaction chro-
matography (HILIC) or porous graphitized carbon (PGC) (Froehlich
et al., 2011; Zauner, Koeleman, Deelder, & Wuhrer, 2010). Recently,
Plomp et al. used trypsin, proteinase K, and chymotrypsin to digest poly-
clonal IgE and were able to determine site-specific assignments and struc-
tural characterization of all six N-linked glycans as a result of the
complementary peptide sequences (Plomp et al., 2013). Schiel et al.
employed extended pronase digestion of RNase B to achieve universal pro-
teolysis and obtain N- and O-linked single amino acid glycans, which were
then permethylated and subjected to MSn analysis (discussed later in this
review) to identify detailed isomeric structure information. This alternative
glycan “release” strategy mitigates some limitations to traditional glycan
cleavage strategies (see below), though peptide sequence and glycosite iden-
tification are compromised (Schiel, Smith, & Phinney, 2013). Hua et al.
were able to achieve site-specific, isomeric, and quantitative glycan profiling
with rapid, in-solution proteinase K, pronase, and subtilisin digestion to
yield short glycopeptides in a strategy called glycoanalytical multispecific
proteolysis (Glyco-AMP) (Hua, Hu, et al., 2013).
z-type peptide fragment ions and observed mass shifts attributed to the
attached glycan (Håkansson et al., 2001). In Fourier transform ion cyclotron
resonance instruments (FT-ICR), soft fragmentation of glycopeptides by
ECD is carried out at low kinetic energy (1.5 eV), resulting in peptide back-
bone fragments (Adamson & Håkansson, 2006). Hot-ECD at moderate
kinetic energy (9 eV) has been applied to permethylated glycans, resulting
in glycosidic and cross-ring fragmentation, and at high kinetic energy
(14 eV), electronic excitation dissociation takes place, resulting in more exten-
sive cross-ring fragmentation to elucidate glycan composition, branching, and
linkage information in detail (Yu, Huang, Lin, & Costello, 2012; Yu et al.,
2013). Other electron-aided fragmentation methods applied to glycopeptides
in negative mode include electron detachment dissociation (Leach et al.,
2012) and negative electron transfer dissociation (Wolff et al., 2010).
Strategies that alternate or combine multiple fragmentation modes have
recently been reported for more complete intact glycopeptide analysis, as
have concerted approaches that separately analyze released glycans and their
deglycosylated peptide counterparts in order to obtain more detailed infor-
mation on each of the parts. Scott et al. employed a strategy of parallel CID,
HCD, and ETD for N-linked glycoproteomic analysis of Campylobacter
jejuni to identify 130 glycopeptides with 75 glycosylation sites following
ZIC-HILIC enrichment of glycopeptides (Scott et al., 2011). Singh et al.
utilized HCD and product ion-triggered ETD fragmentation of N-linked
glycopeptides, based on observed oxonium ions from the initial HCD
MS/MS spectra, to identify glycopeptide sequence, glycan localization,
and glycan structure from RNase B and IgG digests (Singh, Zampronio,
Creese, & Cooper, 2012). Yin et al. compared alternating HCD–ETD
and HCD product ion-triggered ETD MS/MS for analysis of ZIC-HILIC-
enriched derivatized glycopeptides, which were either analyzed directly or
treated with PNGase F in H18 2 O and subjected to separate peptide and glycan
analysis. They found that HCD product ion-triggered ETD detected more
complex/hybrid glycoforms and lower abundant species compared to alter-
nating HCD–ETD, with alternating HCD–ETD selecting larger glycopep-
tides at higher intensities, higher charge, and smaller m/z, resulting in little
overlap of glycopeptide identification between the two acquisition methods.
They also observed little overlap in identifications between direct glycopep-
tide analysis and separate peptide and glycan analysis (Yin et al., 2013). Halim
et al. combined complementary CID-MS2/MS3 and ECD/ETD acquisition
for O-glycan structure characterization, glycopeptide identification, and
O-glycosylation site determination in a nano-LC–MS/MS analysis of
Recent Advances in Glycoproteomics 91
Glycan sequence
CID-MS3
CID-MS2
DVSTPPTVLPDNFPR
y6
b9
ECD-MS2 DVSTPPTVLPDNFPR
M-Ac
c
z1 z3 z4 z7 6
c7 c 8
c3 z8 c10 c11
2.7. Quantitation
The correlation between changes in glycosylation and disease makes relative
quantitative assessment an important part of glycoproteomics studies of dis-
ease states. Relative quantitation of two or more similar samples by MS
requires special considerations in that things like ionization efficiencies, dif-
ferences in sample matrix, instrument to instrument variability, instrument
performance variability, and sample preparation all introduce errors into the
quantitative analysis. In general, MS-based quantitative approaches either
incorporate stable isotope labels onto analytes or are label-free. Label-free
strategies are attractive in that they require little to no modification to a
glycoproteomics workflow, instead relying more heavily on data analysis
and bioinformatics to interpret the semiquantitative value of acquired data
through normalized ion intensities or spectral counting, but generally ought
to be considered a method for screening of potential biomarkers rather
than for definitive quantitative assessment (Orlando, 2013). Isotopic mass
difference labeling strategies enable relative quantitation of a few chemically
derivatized peptide samples within an MS run through comparison of
heavy- and light-labeled peptide ion peak abundances, while isobaric-
labeling strategies involve chemically labeling several peptide samples with
a multiplexed set of isobaric reagents that are indistinguishable in the MS
scan but form discrete reporter ion peaks during MS/MS fragmentation,
enabling relative quantitation based on reporter ion peak abundances
94 Dustin C. Frost and Lingjun Li
(Iliuk, Galan, & Tao, 2008). These approaches require extra sample prepa-
ration by derivatization with costly stable isotope reagents but compensate
for variations in ionization efficiencies and instrument to instrument vari-
ability for coeluting labeled analytes; still, since isotopic labels are incorpo-
rated during sample preparation, losses due to sample preparation can
introduce errors (Orlando, 2013).
Both glycopeptides and glycans can be labeled and quantified using
stable isotope labels. The abundance of enriched glycoproteins can be deter-
mined using established proteomics labeling strategies, like stable isotope
dimethylation (Hsu, Huang, Chow, & Chen, 2003), ICAT (Gygi et al.,
1999), iTRAQ (Ross et al., 2004), or TMT (Thompson et al., 2003) by label-
ing digested glycopeptides, combining samples to be compared, and analyzing
by ESI LC–MS/MS. A study comparing protein-level and peptide-level iso-
baric labeling for serum glycoprotein quantification was recently published by
Nie et al. under the justification that variance in sample preparation prior to
peptide labeling contributes to quantitative error that can be eliminated by
labeling at the protein level. Three commercial isobaric tags for tandem mass
quantitation—iTRAQ 4-plex, TMT 6-plex, and iTRAQ 8-plex—were
compared, optimal solvent conditions for protein labeling were tested, and
several proteases—trypsin, trypsin–GluC, chymotrypsin, and Asp-N—for
digestion of labeled proteins were explored. Under the optimal conditions,
immunodepleted human serum enriched for glycoproteins with AAL LAC
was labeled in 50 mM TEAB:DMSO, combined, digested with trypsin and
Asp-N, deglycosylated with PNGase F, and analyzed via ESI LC–MS/MS
on a Thermo Orbitrap Elite mass spectrometer using HCD fragmentation.
Alternatively, enriched glycoproteins were digested with trypsin prior to
labeling for comparison. The combination of trypsin and Asp-N resulted in
a 30% increase in quantified proteins over the other proteases, and iTRAQ
4-plex gave a 20% increase in identified and quantified proteins over the other
labeling strategies. Protein-level labeling performed slightly better than pep-
tide labeling, with 169 vs. 140 identified proteins and 135 vs. 125 quantified
proteins at a higher quantitative precision (RSD ¼ 11% vs. 15%) (Nie et al.,
2013). Unfortunately, quantitative approaches like this may have limited
implication for disease biomarker studies without glycosylation site or
glycoform analysis. In another study, using bovine fetuin as a model glycopro-
tein for qualitative and quantitative characterization of both glycopeptides and
glycoforms, Ye et al. combined TMT labeling of tryptic glycopeptides and
LC–MS/MS analysis using a Thermo LTQ Orbitrap XL instrument, acquir-
ing tandem mass spectra via alternating CID, HCD, and ETD fragmentation,
Recent Advances in Glycoproteomics 95
infusion and ESI HILIC LC–MS (Wang et al., 2013). Another glycan-
labeling approach incorporates light (12C) and heavy (13C6) hydrazide
biphenyl reagents (4-phenetyl-benzohydrazide; P2GPN) on the reducing
end of glycans, which increases their hydrophobicity and ESI ionization effi-
ciency while avoiding chromatographic shifts (Walker et al., 2011).
Recently, the same authors demonstrated a method, individuality normal-
ization when labeling with isotopic glycan hydrazide tags (INLIGHT), to
compensate for quantitative inaccuracies due to overlapping isotopic enve-
lopes of light and heavy P2GPN-labeled species (Walker, Taylor, &
Muddiman, 2013). An isobaric stable isotope labeling reagent, glyco-
TMT, was recently reported for mass difference or isobaric tandem mass
quantitation of glycans (Fig. 3.4). The reagent consists of an isotopic reporter
group and a mass balance group and bears a carbonyl-reactive aminooxy
group for reaction with the glycan reducing end. Light (TMT0) and heavy
(TMT6) amino oxy labels were demonstrated to have an accessible dynamic
range of 1:20 by MALDI-TOF analysis of N-glycans from ovalbumin. The
isobaric versions TMT6-128 and TMT6-131, which instead yield discrete
reporter ions in the low mass region upon tandem mass fragmentation,
do not add spectral complexity to the parent scan, but were demonstrated
to show limited dynamic range by MALDI-TOF/TOF analysis compared
to the mass difference labels, though the choice of instrumentation was likely
the limiting factor (Hahne et al., 2012). Gong et al. recently demonstrated
duplex labeling of N-glycans from a monoclonal antibody with commer-
cially available amine-reactive TMT0 and TMT6. Under basic conditions
(pH 8.3) in 50 mmol TEAB buffer, PNGase F release of N-glycans results
in stable N-glycosylamines which are readily labeled by the NHS-ester of
TMT reagents. The dynamic range for three coeluting TMT6:TMT0-
labeled glycans was shown to be accurate to 1:20 by LC–MS/MS analysis
on a Thermo LTQ Orbitrap XL (Gong et al., 2013). Detailed reviews of
glycan-labeling strategies for quantitative glycomics have been recently pub-
lished elsewhere (Mechref, Hu, Desantos-Garcia, Hussein, & Tang, 2013;
Ruhaak et al., 2010).
Once candidate biomarkers have been determined and sufficiently isolated
from the complex sample, targeted, label-free MS quantitation can be per-
formed via selected or multiple reaction monitoring (SRM or MRM) using
a triple quadrupole (QqQ), Q-Trap, or hybrid QqTOF mass spectrometers.
In this approach, MS analysis time is focused exclusively on analytes of interest
and all others are excluded in order to increase sensitivity by several orders of
magnitude. This is achieved by isolating precursor ions of interest,
Recent Advances in Glycoproteomics 97
A
Glyco-TMT 5 Da
*
* Oxime formation
*
*
*
*
Reporter Mass normalizer Aminooxy
B
1553.715
Man5GlcNAc2
1553.630 1715.700
5 Da
2041.870 TMT 0 TMT 6
1432.603
1635.694 2448.059
1959.815
2610.135
C
Y4
Y3a
6 6 Y2 Y1
TMT -128 TMT -131
128.1 131.1 TMT
Y4 B4
0,2
1198.7 A5
B3 B4
Y3b 1318.7
C4
Y4
1216.7
1011.5
B3 Man6GlcNAc2-TMT
C3 1416.8
995.3 1720.8
+
Y1(H ) Y1 Hex4 1013.5 TMT
523.3 545.3 671.3 B4/Y4
+ Metastable decay
Hex(H ) Hex2(H )
+ 1036.4
626.4
163.1 325.1 3,5
+
A4
GlcNAc(H ) Y2 1069.5
204.1 401.2 748.3
Figure 3.4 (A) Structure of Glyco-TMT reagent with reporter group, mass normalizer,
and aminooxy functional group. Locations of isotopic substitution in the Glyco-TMT
reporter structure are indicated (*). A mass shift of 5 Da is incorporated between
heavy- and light-labeled glycans; the heavy-labeled species in the spectra is indicated
(*). (B) MALDI-TOF MS1 level quantitation of heavy- and light-labeled (TMT0/TMT6 1:1)
N-glycans from ovalbumin. (C) MALDI-TOF/TOF MS2 level reporter ion-based quantita-
tion and structural determination of isobarically labeled N-linked ovalbum glycan Man6-
GlcNAc2 (m/z 1720.708, aminooxy TMT6-128/131, 1:1). Circle, Man; square, GlcNAc;
diamond, NeuNAc. Adapted with permission from Hahne et al. (2012). Copyright 2012
American Chemical Society.
98 Dustin C. Frost and Lingjun Li
fragmenting them, and selecting specific product fragment ions for detection
and scheduling precursor/product “transitions” based on retention times over
an LC run. Absolute quantitation is accomplished by spiking synthetic, stable
isotope-labeled versions of each analyte peptide in known concentrations and
comparing signal intensities of the analyte and stable isotope reference to
determine protein concentration (Gillette & Carr, 2013). Recent glyco-
proteomics studies have employed MRM approaches for quantification of
core fucosylated peptides in hepatocellular carcinoma (HCC) patient serum
(Zhao et al., 2011), sialylated peptides from mouse serum (Kurogochi
et al., 2010), IgG glycopeptides and their site-specific glycoform abundances
in serum (Hong, Lebrilla, Miyamoto, & Ruhaak, 2013), haptoglobin glyco-
peptides and their site-specific glycoform abundances in liver disease (Sanda
et al., 2013), and serum glycopeptides based on selection of oxonium ion tran-
sitions rather than peptide ion transitions (Song, Pyreddy, & Mechref, 2012).
Targeted MS-based quantitation by MRM is seeing wide clinical and
bioanalytical use in biomarker studies and has been reviewed extensively
(Boja & Rodriguez, 2012; Gillette & Carr, 2013; Kitteringham, Jenkins,
Lane, Elliott, & Park, 2009; Lemoine et al., 2012; Liebler & Zimmerman,
2013; Meng & Veenstra, 2011; Percy, Parker, & Borchers, 2013).
A recently developed alternative method of label-free quantitation is
SWATH-MS, a data-independent acquisition approach that rapidly cycles
through defined 25-Da precursor isolation windows (swaths) to obtain
broad fragmentation data for all analytes using Q-TOF, QqTOF, or triple
TOF mass spectrometers. This technique has been shown to provide qual-
itative and quantitative performance comparable to SRM (Gillet et al.,
2012). Aebersold and coworkers used SWATH-MS for quantitative analysis
of N-linked glycoproteins in human plasma and observed similar quantita-
tive accuracy, reproducibility, and dynamic range between SWATH-MS
and SRM, though SRM was the more sensitive approach (Liu et al., 2013).
2.8. Bioinformatics
Following MS-based glycoproteomics analyses, the acquired tandem mass
spectral data contains a wealth of information that must be interpreted using
bioinformatics software. Such tools determine protein identifications based
on sequencing of peptide fragment ions and protein database search, glyco-
sylation site assignment based on peptide fragment ions containing markers
of glycosylation (e.g., Asn->Asp conversion following PNGase F treatment,
N-linked GlcNAc, retained glycans from ETD), and glycan characterization
Recent Advances in Glycoproteomics 99
based on oxonium ions, glycosidic cleavage ions, and cross-ring cleavage ions
and glycan structural database matching. Given the vast amounts of data
acquired during large-scale glycoproteomics experiments, the importance
of bioinformatics in the glycoproteomics workflow cannot be overstated.
While a detailed summary of current bioinformatics tools is beyond the scope
of this review, several recent reviews are available for further information
(Dallas, Martin, Hua, & German, 2013; Li, Glinskii, & Glinsky, 2013;
Woodin, Maxon, & Desaire, 2013). Recent bioinformatics tools introduced
since the publication of these reviews include: SweetSEQer, a simple,
open source tool for de novo analysis of glycoconjugate MS/MS spectra with
annotations (Serang et al., 2013); GlycoFragwork, a framework for
N-glycopeptide scoring and glycan sequencing that combines LC–MS/MS
data set alignment, scoring of CID, HCD, and ETD spectra, and an algorithm
for elucidating glycan structure based on peaks in the CID spectrum with
scoring, ranking, and FDR reporting for potential glycans (Mayampurath
et al., 2014); PTM MarkerFinder, a tool for screening database search output
of HCD and ETD data for marker ions of glycosylation (e.g., HexNAc, Hex)
(Nanni et al., 2013); GlycoPep Detector, a tool for assigning glycopeptide
composition based on ETD spectra (Zhu, Hua, Clark, Go, & Desaire,
2013); and SRMAtlas, a library of SRM assays of N-glycosites for targeted
quantitative analyses of N-glycoprotein biomarkers (Hüttenhain et al., 2013).
colon cancer, pancreatic cancer, lung cancer, and liver cancer. Several can-
cer biomarkers have been identified: prostate-specific antigen for prostate
cancer; CA125 and sialylated LewisX glycans for ovarian cancer; HER2,
CA27-29, and O-glycan sialylation of CA15-3, and sialylated LewisX gly-
cans for breast cancer; carcinoembryonic antigen for colon cancer;
CA19-9 and fucosylated haptoglobin for pancreatic cancer; sialylated
LewisX glycans for lung cancer; alpha-fetoprotein (AFP) and AFP-L3 core
fucosylation for HCC (Adamczyk, Struwe, Ercan, Nigrovic, & Rudd, 2013;
Kuzmanov, Kosanam, & Diamandis, 2013; Mechref et al., 2012). In a recent
study by Hua et al., chip-based PGC nano-LC-TOF/MS was used to quan-
titatively profile N-glycans from 15 different cancer cell lines isolated from
ovarian, breast, lung, cervical, and lymphatic cancer patients to uncover sig-
nificant relative abundance changes in broad glycan classes (high mannose,
complex/hybrid fucosylated, complex/hybrid sialylated, etc.) and individual
glycan structures to differentiate between cell lines (Figs. 3.5 and 3.6)
(Hua et al., 2014). Once cancer-associated glycoforms are identified,
´104
5
Intensity (counts)
0
2 3 4 5 6 7 8 9 10
Retention time (min)
100 1.0
Lymphoma
Correlation (R)
i
aj
R
Ovarian carcinoma
os
am
R
9
92
75
I-H
-1
C
Relative abundance (%)
BL
N
BC
0.0
-2
ES
50
25
0
High man C/H C/H-F C/H-S C/H-FS
N-glycan type and decoration
Figure 3.6 Glycan class profiles of four B-cell lymphoma cell lines (Raji, Ramos, NCI-H929,
and BCBL-1) and one ovarian carcinoma cell line (ES-2). Relative abundances are shown
for each glycan biosynthetic class. Inset: color-coded representation of the Pearson cor-
relation coefficient (R) between each pair of cell lines, ranging from red (high correlation;
gray top of scale in print version) to blue (low correlation; black bottom of scale with hash
lines in print version), along with hierarchical clustering trees. Adapted with permission
from Hua et al. (2014). Copyright 2014 American Chemical Society.
glycoproteins with fucose and sialic acid from several luminal (less aggres-
sive) and triple negative (more aggressive) breast cancer cell lines, followed
by deglycosylation with PNGase F and LC–MS/MS analysis on a Thermo
LTQ Orbitrap XL. Their statistical analysis of over 1000 glycosites on 533
identified glycoproteins revealed that 100 glycosites were solely detected in
several triple negative lines, and differential expression of fucosyl- and
sialyltransferases between the two line subtypes suggests that changes in gly-
cosylation may act to indicate putative biomarkers (Drake et al., 2012). Ahn
et al. also used AAL to enrich fucosylated glycoproteins from small-cell lung
cancer (SCLC) patient sera samples. A combination of label-free and
iTRAQ-labeled quantitative MS analysis revealed decreases in abundance
of serum paraoxonase (PON1) alongside increases in the degree of
N-linked glycan fucosylation of PON1 (Ahn, Sung, et al., 2013), findings
which support previous reports of increased fucosylation and sialylation of
serum PON1 in liver cirrhosis patients (Sun et al., 2012) and suggest
PON1 glycosylation patterns as a biomarker for both HCC and SCLC.
IMS-MS can uniquely facilitate MS-based cancer biomarker glyco-
proteomics research due to its ability to separate glycan isomers during MS
analysis, prior to detection. Isaiolovic et al. demonstrated the potential of
IMS-MS for cancer biomarker discovery by characterizing serum N-linked
glycans from patients with cirrhosis of the liver and liver cancer patients.
The combination of supervised principal component analysis (PCA) with
ion mobility distributions of glycan isomers and conformers of 10 different
glycans was sufficient to discriminate cirrhosis and liver cancer from healthy
patients (Isailovic et al., 2012). The same IMS–MS and PCA approach of
serum N-glycan analysis was then used for distinguishing phenotypes of
Barrett’s esophagus, high-grade dysplasia, and esophageal adenocarcinoma
from normal control. Composite ion mobility distributions constructed from
11 glycans revealed 46 features that allowed unambiguous differentiation of
the esophageal adenocarcinoma samples from the normal control samples;
the authors noted, however, that improvements in IMS separation efficiency
are required to observe individual isomer contributions (Gaye et al., 2012).
Abnormal or incomplete O-glycosylation has been recognized as a bio-
marker for cancer and is commonly observed in carcinomas. Mucins and
glycoproteins in tumor cells feature truncation of O-glycans to GalNAc-
a-Ser/Thr, also known as the Tn antigen, which does not appear in normal
tissues. It has been reported that 70–90% of cancers of the colon, lung, bladder,
cervix, ovary, stomach, and prostate express this O-glycan truncation, and in
other cancers, the expression correlates with poor prognosis and metastasis
Recent Advances in Glycoproteomics 103
( Ju et al., 2013). O-GlcNAc has also been implicated in the cause and pro-
gression of cancer (Fardini, Dehennaut, Lefebvre, & Issad, 2013). The targeted
enrichment of O-linked GalNAc or GlcNAc has been demonstrated recently
using the Helix pomotia agglutinin lectin with human breast cancer cell lines
(Rambaruth, Greenwell, & Dwek, 2012), and O-GlcNAc enrichment by
reversible hydrazide chemistry, mentioned previously, has also been devel-
oped (Nishikaze et al., 2013). The identification of O-GlcNAc sites by
MS/MS has been facilitated appreciably by ETD fragmentation, as demon-
strated by several recent studies (Chalkley, Thalhammer, Schoepfer, &
Burlingame, 2009; Myers, Daou, Affar, & Burlingame, 2013; Wang et al.,
2010). Current reviews have covered O-GlcNAcylation and its role in disease
and cancer in detail (Copeland et al., 2013; Fardini et al., 2013; Hart, Slawson,
Ramirez-Correa, & Lagerlof, 2011).
Targeted analysis of glycoprotein cancer biomarker candidates has been
performed using SRM and MRM approaches. Yoo and coworkers have
investigated aberrant glycoforms of tissue inhibitor of metalloproteinase
1 (TIMP1), a colorectal cancer (CRC) biomarker candidate, in serum
and from CRC cell lines that combine L-PHA lectin enrichment, stable
isotope standard and capture by antipeptide antibody of a target tryptic
peptide, and quantitative MRM analysis (Ahn et al., 2010, 2009). Abun-
dance of the aberrant glycoform TIMP1 in CRC cells was shown to be
11.7-fold greater than control (Ahn et al., 2010). The same researchers
recently used AAL enrichment and MRM quantitation for aberrant protein
glycosylation analysis of a-1-antitrypsin and a-2HS-glycoprotein in HCC
plasma, observing a 4.7- and 2.2-fold increase, respectively, in the aberrant
glycoforms (Ahn, Shin, et al., 2013). Sanda et al. used MRM to study aber-
rant hyperfucosylation of a haptoglobin N-glycosite in HCC and cirrhosis
patient plasma, monitoring oxonium ions for quantitation. Relative quan-
titation among HCC, cirrhosis, and control revealed that glycoforms dis-
playing multiple outer arm fucose were considerably elevated in HCC and
cirrhosis (Sanda et al., 2013).
Cancer has become a primary focus of MS-based glycoproteomics and
glycomics biomarker studies. A comprehensive summary of the highly
active field is beyond the scope of this review, and interested readers are
encouraged to consult the numerous published reviews covering recent
research progress (Adamczyk et al., 2013; Fardini et al., 2013; Kim &
Misek, 2011; Kuzmanov et al., 2013; Lin et al., 2012; Meany & Chan,
2011; Mechref et al., 2012; Ruhaak, Miyamoto, & Lebrilla, 2013; Tan,
Lee, & Chung, 2012; Ueda, 2013).
104 Dustin C. Frost and Lingjun Li
4. CONCLUDING REMARKS
MS-based glycoproteomics and glycomics have become a vital part of
biomedical research for discovery and characterization of disease biomarkers
due to rapid technological advances in affinity enrichment, chromatographic
Recent Advances in Glycoproteomics 107
ACKNOWLEDGMENTS
Preparation of this manuscript was supported in part by the National Institutes of Health
Grant R01 NS071513. L. L. acknowledges an H. I. Romnes Faculty Fellowship.
REFERENCES
Adamczyk, B., Struwe, W. B., Ercan, A., Nigrovic, P. A., & Rudd, P. M. (2013). Charac-
terization of fibrinogen glycosylation and its importance for serum/plasma N-glycome
analysis. Journal of Proteome Research, 12(1), 444–454. http://dx.doi.org/10.1021/
pr300813h.
Adamson, J. T., & Håkansson, K. (2006). Infrared multiphoton dissociation and electron cap-
ture dissociation of high-mannose type glycopeptides. Journal of Proteome Research, 5(3),
493–501. http://dx.doi.org/10.1021/pr0504081.
Ahn, Y. H., Kim, Y.-S., Ji, E. S., Lee, J. Y., Jung, J.-A., Ko, J. H., et al. (2010). Comparative
quantitation of aberrant glycoforms by lectin-based glycoprotein enrichment coupled
with multiple-reaction monitoring mass spectrometry. Analytical Chemistry, 82(11),
4441–4447. http://dx.doi.org/10.1021/ac1001965.
Ahn, Y. H., Lee, J. Y., Lee, J. Y., Kim, Y.-S., Ko, J. H., & Yoo, J. S. (2009). Quantitative
analysis of an aberrant glycoform of TIMP1 from colon cancer serum by L-PHA-
enrichment and SISCAPA with MRM mass spectrometry. Journal of Proteome Research,
8(9), 4216–4224. http://dx.doi.org/10.1021/pr900269s.
Ahn, Y. H., Shin, P. M., Kim, Y.-S., Oh, N. R., Ji, E. S., Kim, K. H., et al. (2013). Quan-
titative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-
enrichment and MRM mass spectrometry. The Analyst, 138(21), 6454–6462. http://
dx.doi.org/10.1039/c3an01126g.
Ahn, J.-M., Sung, H.-J., Yoon, Y.-H., Kim, B.-G., Yang, W. S., Lee, C., et al. (2013). Inte-
grated glycoproteomics demonstrates fucosylated serum paraoxonase 1 alterations in
small cell lung cancer. Molecular & Cellular Proteomics. http://dx.doi.org/10.1074/mcp.
M113.028621.
108 Dustin C. Frost and Lingjun Li
Alley, W. R., Mann, B. F., & Novotny, M. V. (2013). High-sensitivity analytical approaches
for the structural characterization of glycoproteins. Chemical Reviews, 113(4), 2668–2732.
http://dx.doi.org/10.1021/cr3003714.
Alley, W. R., Mechref, Y., & Novotny, M. V. (2009a). Characterization of glycopeptides by
combining collision-induced dissociation and electron-transfer dissociation mass spec-
trometry data. Rapid Communications in Mass Spectrometry, 23(1), 161–170. http://dx.
doi.org/10.1002/rcm.3850.
Alley, W. R., Mechref, Y., & Novotny, M. V. (2009b). Use of activated graphitized carbon
chips for liquid chromatography/mass spectrometric and tandem mass spectrometric
analysis of tryptic glycopeptides. Rapid Communications in Mass Spectrometry, 23(4),
495–505. http://dx.doi.org/10.1002/rcm.3899.
Alvarez-Manilla, G., Atwood, J., Guo, Y., Warren, N. L., Orlando, R., & Pierce, M. (2006).
Tools for glycoproteomic analysis: Size exclusion chromatography facilitates identifica-
tion of tryptic glycopeptides with N-linked glycosylation sites. Journal of Proteome
Research, 5(3), 701–708. http://dx.doi.org/10.1021/pr050275j.
Alvarez-Manilla, G., Warren, N. L., Abney, T., Atwood, J., Azadi, P., York, W. S., et al.
(2007). Tools for glycomics: Relative quantitation of glycans by isotopic permethylation
using 13CH3I. Glycobiology, 17(7), 677–687. http://dx.doi.org/10.1093/glycob/cwm033.
Alvarez-Manilla, G., Warren, N. L., Atwood, J., III, Orlando, R., Dalton, S., & Pierce, M.
(2010). Glycoproteomic analysis of embryonic stem cells: Identification of potential
glycobiomarkers using lectin affinity chromatography of glycopeptides. Journal of Prote-
ome Research, 9(5), 2062–2075. http://dx.doi.org/10.1021/pr8007489.
An, H. J., Froehlich, J. W., & Lebrilla, C. B. (2009). Determination of glycosylation sites and
site-specific heterogeneity in glycoproteins. Current Opinion in Chemical Biology, 13(4),
421–426. http://dx.doi.org/10.1016/j.cbpa.2009.07.022.
Anderson, N. L., & Anderson, N. G. (2002). The human plasma proteome: History, char-
acter, and diagnostic prospects. Molecular & Cellular Proteomics, 1(11), 845–867. http://dx.
doi.org/10.1074/mcp.R200007.
Apweiler, R., Hermjakob, H., & Sharon, N. (1999). On the frequency of protein glycosyl-
ation, as deduced from analysis of the SWISS-PROT database. Biochimica et Biophysica
Acta, 1473(1), 4–8. http://dx.doi.org/10.1016/s0304-4165(99)00165-8.
Atwood, J. A., Cheng, L., Alvarez-Manilla, G., Warren, N. L., York, W. S., & Orlando, R.
(2008). Quantitation by isobaric labeling: Applications to glycomics. Journal of Proteome
Research, 7(1), 367–374. http://dx.doi.org/10.1021/pr070476i.
Atwood, J. A., Sahoo, S. S., Alvarez-Manilla, G., Weatherly, D. B., Kolli, K., Orlando, R.,
et al. (2005). Simple modification of a protein database for mass spectral identification of
N-linked glycopeptides. Rapid Communications in Mass Spectrometry, 19(21), 3002–3006.
http://dx.doi.org/10.1002/rcm.2162.
Barone, R., Sturiale, L., Palmigiano, A., & Zappia, M. (2012). Glycomics of pediatric and
adulthood diseases of the central nervous system. Journal of Proteomics, 75(17), 5123–5139.
http://dx.doi.org/10.1016/j.jprot.2012.07.007.
Berven, F. S., Ahmad, R., Clauser, K. R., & Carr, S. A. (2010). Optimizing performance of
glycopeptide capture for plasma proteomics. Journal of Proteome Research, 9(4),
1706–1715. http://dx.doi.org/10.1021/pr900845m.
Boja, E. S., & Rodriguez, H. (2012). Mass spectrometry-based targeted quantitative prote-
omics: Achieving sensitive and reproducible detection of proteins. Proteomics, 12(8),
1093–1110. http://dx.doi.org/10.1002/pmic.201100387.
Botella-López, A., Burgaya, F., Gavı́n, R., Garcı́a-Ayllón, M. S., Gómez-Tortosa, E., Peña-
Casanova, J., et al. (2006). Reelin expression and glycosylation patterns are altered in
Alzheimer’s disease. Proceedings of the National Academy of Sciences of the United States of
America, 103(14), 5573–5578. http://dx.doi.org/10.1073/pnas.0601279103.
Recent Advances in Glycoproteomics 109
€
Brinkmalm, G., Portelius, E., Ohrfelt, A., Mattsson, N., Persson, R., Gustavsson, M. K.,
et al. (2012). An online nano-LC-ESI-FTICR-MS method for comprehensive charac-
terization of endogenous fragments from amyloid b and amyloid precursor protein in
human and cat cerebrospinal fluid. Journal of Mass Spectrometry, 47(5), 591–603. http://
dx.doi.org/10.1002/jms.2987.
Browning, S., Baker, C. A., Smith, E., Mahal, S. P., Herva, M. E., Demczyk, C. A., et al.
(2011). Abrogation of complex glycosylation by swainsonine results in strain- and cell-
specific inhibition of prion replication. Journal of Biological Chemistry, 286(47),
40962–40973. http://dx.doi.org/10.1074/jbc.M111.283978.
Carlson, D. M. (1968). Structures and immunochemical properties of oligosaccharides iso-
lated from pig submaxillary mucins. Journal of Biological Chemistry, 243(3), 616–626.
Chalkley, R. J., Thalhammer, A., Schoepfer, R., & Burlingame, A. L. (2009). Identification
of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry
on native peptides. Proceedings of the National Academy of Sciences of the United States of
America, 106(22), 8894–8899. http://dx.doi.org/10.1073/pnas.0900288106.
Chen, R., Jiang, X., Sun, D., Han, G., Wang, F., Ye, M., et al. (2009). Glycoproteomics
analysis of human liver tissue by combination of multiple enzyme digestion and hydra-
zide chemistry. Journal of Proteome Research, 8(2), 651–661. http://dx.doi.org/10.1021/
pr8008012.
Chen, J., Shah, P., & Zhang, H. (2013). Solid phase extraction of N-linked glycopeptides
using hydrazide tip. Analytical Chemistry, 85(22), 10670–10674. http://dx.doi.org/
10.1021/ac401812b.
Chen, C. C., Su, W. C., Huang, B. Y., Chen, Y. J., & Tai, H. C. (2014). Interaction modes
and approaches to glycopeptide and glycoprotein enrichment. The Analyst, 139(4),
688–704. http://dx.doi.org/10.1039/c3an01813j.
Cho, W., Jung, K., & Regnier, F. E. (2008). Use of glycan targeting antibodies to identify
cancer-associated glycoproteins in plasma of breast cancer patients. Analytical Chemistry,
80(14), 5286–5292. http://dx.doi.org/10.1021/ac8008675.
Choi, E., Loo, D., Dennis, J. W., O’Leary, C. A., & Hill, M. M. (2011). High-throughput
lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein bio-
marker discovery. Electrophoresis, 32(24), 3564–3575. http://dx.doi.org/10.1002/
elps.201100341.
Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the permethylation of
carbohydrates. Carbohydrate Research, 131, 209–217. http://dx.doi.org/10.1016/0008-
6215(84)85242-8.
Clowers, B. H., Dodds, E. D., Seipert, R. R., & Lebrilla, C. B. (2007). Site determination of
protein glycosylation based on digestion with immobilized nonspecific proteases and
Fourier transform ion cyclotron resonance mass spectrometry. Journal of Proteome
Research, 6(10), 4032–4040. http://dx.doi.org/10.1021/pr070317z.
Comer, F. I., Vosseller, K., Wells, L., Accavitti, M. A., & Hart, G. W. (2001). Characterization
of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Analytical
Biochemistry, 293(2), 169–177. http://dx.doi.org/10.1006/abio.2001.5132.
Copeland, R. J., Han, G., & Hart, G. W. (2013). O-GlcNAcomics—Revealing roles of
O-GlcNAcylation in disease mechanisms and development of potential diagnostics. Pro-
teomics. Clinical Applications, 7, 597–606. http://dx.doi.org/10.1002/prca.201300001.
Craft, G. E., Chen, A., & Nairn, A. C. (2013). Recent advances in quantitative
neuroproteomics. Methods, 61(3), 186–218. http://dx.doi.org/10.1016/j.ymeth.2013.
04.008.
Creese, A. J., & Cooper, H. J. (2012). Separation and identification of isomeric glycopeptides
by high field asymmetric waveform ion mobility spectrometry. Analytical Chemistry,
84(5), 2597–2601. http://dx.doi.org/10.1021/ac203321y.
110 Dustin C. Frost and Lingjun Li
Gaye, M. M., Valentine, S. J., Hu, Y., Mirjankar, N., Hammoud, Z. T., Mechref, Y., et al.
(2012). Ion mobility-mass spectrometry analysis of serum N-linked glycans from esoph-
ageal adenocarcinoma phenotypes. Journal of Proteome Research, 11(12), 6102–6110.
http://dx.doi.org/10.1021/pr300756e.
Gbormittah, F. O., Haab, B. B., Partyka, K., Garcia-Ott, C., Hincapie, M., &
Hancock, W. S. (2013). Characterization of glycoproteins in pancreatic cyst fluid using
a high performance multiple lectin affinity chromatography platform. Journal of Proteome
Research. http://dx.doi.org/10.1021/pr400813u.
Gerlach, J. Q., Kilcoyne, M., Farrell, M. P., Kane, M., & Joshi, L. (2012). Differential release
of high mannose structural isoforms by fungal and bacterial endo-b-N-acety-
lglucosaminidases. Molecular BioSystems, 8(5), 1472. http://dx.doi.org/10.1039/
c2mb05455h.
Gillet, L. C., Navarro, P., Tate, S., R€ost, H., Selevsek, N., Reiter, L., et al. (2012). Targeted
data extraction of the MS/MS spectra generated by data-independent acquisition: A new
concept for consistent and accurate proteome analysis. Molecular & Cellular Proteomics.
11(6). http://dx.doi.org/10.1074/mcp.O111.016717, O111.016717.
Gillette, M. A., & Carr, S. A. (2013). Quantitative analysis of peptides and proteins in bio-
medicine by targeted mass spectrometry. Nature Methods, 10(1), 28–34.
Goetz, J. A., Novotny, M. V., & Mechref, Y. (2009). Enzymatic/chemical release of
O-glycans allowing MS analysis at high sensitivity. Analytical Chemistry, 81(23),
9546–9552. http://dx.doi.org/10.1021/ac901363h.
Gong, B., Hoyt, E., Lynaugh, H., Burnina, I., Moore, R., Thompson, A., et al. (2013). N-
glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative anal-
ysis of N-linked glycans. Analytical and Bioanalytical Chemistry, 405(17), 5825–5831.
http://dx.doi.org/10.1007/s00216-013-6988-9.
Grass, J., Pabst, M., Chang, M., Wozny, M., & Altmann, F. (2011). Analysis of recombinant
human follicle-stimulating hormone (FSH) by mass spectrometric approaches. Analytical
and Bioanalytical Chemistry, 400, 2427–2438. http://dx.doi.org/10.1007/s00216-011-
4923-5.
Guillard, M., Gloerich, J., Wessels, H. J. C. T., Morava, E., Wevers, R. A., & Lefeber, D. J.
(2009). Automated measurement of permethylated serum N-glycans by MALDI-linear
ion trap mass spectrometry. Carbohydrate Research, 344(12), 1550–1557. http://dx.doi.
org/10.1016/j.carres.2009.06.010.
Gupta, G., Surolia, A., & Sampathkumar, S.-G. (2010). Lectin microarrays for glycomic anal-
ysis. OMICS: A Journal of Integrative Biology, 14(4), 419–436. http://dx.doi.org/10.1089/
omi.2009.0150.
Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., & Aebersold, R. (1999). Quan-
titative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Bio-
technology, 17(10), 994–999. http://dx.doi.org/10.1038/13690.
Hage, D. S., Anguizola, J. A., Bi, C., Li, R., Matsuda, R., Papastavros, E., et al. (2012). Phar-
maceutical and biomedical applications of affinity chromatography: Recent trends and
developments. Journal of Pharmaceutical and Biomedical Analysis, 69, 93–105. http://dx.
doi.org/10.1016/j.jpba.2012.01.004.
Hägglund, P., Matthiesen, R., Elortza, F., Højrup, P., Roepstorff, P., Jensen, O. N., et al.
(2007). An enzymatic deglycosylation scheme enabling identification of core fucosylated
N-glycans and O-glycosylation site mapping of human plasma proteins. Journal of Prote-
ome Research, 6(8), 3021–3031. http://dx.doi.org/10.1021/pr0700605.
Hahne, H., Neubert, P., Kuhn, K., Etienne, C., Bomgarden, R., Rogers, J. C., et al. (2012).
Carbonyl-reactive tandem mass tags for the proteome-wide quantification of N-linked
glycans. Analytical Chemistry, 84(8), 3716–3724. http://dx.doi.org/10.1021/ac300197c.
Håkansson, K., Cooper, H. J., Emmett, M. R., Costello, C. E., Marshall, A. G., &
Nilsson, C. L. (2001). Electron capture dissociation and infrared multiphoton dissocia-
tion MS/MS of an N-glycosylated tryptic peptide to yield complementary sequence
112 Dustin C. Frost and Lingjun Li
Kaji, H., Saito, H., Yamauchi, Y., Shinkawa, T., Taoka, M., Hirabayashi, J., et al. (2003).
Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify
N-linked glycoproteins. Nature Biotechnology, 21(6), 667–672. http://dx.doi.org/
10.1038/nbt829.
Kim, E. H., & Misek, D. E. (2011). Glycoproteomics-based identification of cancer bio-
markers. International Journal of Proteomics, 2011, 601937. http://dx.doi.org/
10.1155/2011/601937.
Kim, C., Nam, D. W., Park, S. Y., Song, H., Hong, H. S., Boo, J. H., et al. (2013). O-linked
b-N-acetylglucosaminidase inhibitor attenuates b-amyloid plaque and rescues memory
impairment. Neurobiology of Aging, 34(1), 275–285. http://dx.doi.org/10.1016/j.
neurobiolaging.2012.03.001.
Kitteringham, N. R., Jenkins, R. E., Lane, C. S., Elliott, V. L., & Park, B. K. (2009).
Multiple reaction monitoring for quantitative biomarker analysis in proteomics and
metabolomics. Journal of Chromatography. B, Analytical Technologies in the Biomedical
and Life Sciences, 877(13), 1229–1239. http://dx.doi.org/10.1016/j.jchromb.
2008.11.013.
Klement, E., Lipinszki, Z., Kupihár, Z., Udvardy, A., & Medzihradszky, K. F. (2010).
Enrichment of O-GlcNAc modified proteins by the periodate oxidationhydrazide
resin capture approach. Journal of Proteome Research, 9(5), 2200–2206. http://dx.doi.
org/10.1021/pr900984h.
Kozak, R. P., Royle, L., Gardner, R. A., Fernandes, D. L., & Wuhrer, M. (2012). Suppres-
sion of peeling during the release of O-glycans by hydrazinolysis. Analytical Biochemistry,
423(1), 119–128. http://dx.doi.org/10.1016/j.ab.2012.01.002.
Kullolli, M., Hancock, W. S., & Hincapie, M. (2008). Preparation of a high-performance
multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human
plasma glycoproteins. Journal of Separation Science, 31(14), 2733–2739. http://dx.doi.org/
10.1002/jssc.200800233.
Kullolli, M., Hancock, W. S., & Hincapie, M. (2010). Automated platform for fractionation
of human plasma glycoproteome in clinical proteomics. Analytical Chemistry, 82(1),
115–120. http://dx.doi.org/10.1021/ac9013308.
Kurogochi, M., Matsushista, T., Amano, M., Furukawa, J.-I., Shinohara, Y., Aoshima, M.,
et al. (2010). Sialic acid-focused quantitative mouse serum glycoproteomics by multiple
reaction monitoring assay. Molecular & Cellular Proteomics, 9(11), 2354–2368. http://dx.
doi.org/10.1074/mcp.M110.000430.
Küster, B., & Mann, M. (1999). 18O-labeling of N-glycosylation sites to improve the iden-
tification of gel-separated glycoproteins using peptide mass mapping and database
searching. Analytical Chemistry, 71(7), 1431–1440. http://dx.doi.org/10.1021/
ac981012u.
Kuzmanov, U., Kosanam, H., & Diamandis, E. P. (2013). The sweet and sour of serological
glycoprotein tumor biomarker quantification. BMC Medicine, 11, 31. http://dx.doi.org/
10.1186/1741-7015-11-31.
Larsen, M. R., Jensen, S. S., Jakobsen, L. A., & Heegaard, N. H. H. (2007). Exploring the
sialiome using titanium dioxide chromatography and mass spectrometry. Molecular &
Cellular Proteomics, 6(10), 1778–1787. http://dx.doi.org/10.1074/mcp.M700086-
MCP200.
Leach, F. E., Ly, M., Laremore, T. N., Wolff, J. J., Perlow, J., Linhardt, R. J., et al. (2012).
Hexuronic acid stereochemistry determination in chondroitin sulfate glycosaminoglycan
oligosaccharides by electron detachment dissociation. Journal of the American Society for
Mass Spectrometry, 23(9), 1488–1497. http://dx.doi.org/10.1007/s13361-012-0428-5.
Lemoine, J., Fortin, T., Salvador, A., Jaffuel, A., Charrier, J.-P., & Choquet-Kastylevsky, G.
(2012). The current status of clinical proteomics and the use of MRM and MRM(3) for
Recent Advances in Glycoproteomics 115
Meany, D. L., & Chan, D. W. (2011). Aberrant glycosylation associated with enzymes as
cancer biomarkers. Clinical Proteomics, 8(1), 7. http://dx.doi.org/10.1186/1559-0275-
8-7.
Mechref, Y., Hu, Y., Desantos-Garcia, J. L., Hussein, A., & Tang, H. (2013). Quantitative
glycomics strategies. Molecular & Cellular Proteomics, 12(4), 874–884. http://dx.doi.org/
10.1074/mcp.R112.026310.
Mechref, Y., Hu, Y., Garcia, A., Zhou, S., Desantos-Garcia, J. L., & Hussein, A. (2012).
Defining putative glycan cancer biomarkers by MS. Bioanalysis, 4(20), 2457–2469.
http://dx.doi.org/10.4155/bio.12.246.
Mechref, Y., & Novotny, M. V. (2002). Structural investigations of glycoconjugates at high
sensitivity. Chemical Reviews, 102(2), 321–370. http://dx.doi.org/10.1021/cr0103017.
Medvedev, A., Kopylov, A., Buneeva, O., Zgoda, V., & Archakov, A. (2012). Affinity-based
proteomic profiling: Problems and achievements. Proteomics, 12(4–5), 621–637. http://
dx.doi.org/10.1002/pmic.201100373.
Meng, Z., & Veenstra, T. D. (2011). Targeted mass spectrometry approaches for protein bio-
marker verification. Journal of Proteomics, 74(12), 2650–2659. http://dx.doi.org/10.1016/
j.jprot.2011.04.011.
Miyoshi, E., Moriwaki, K., & Nakagawa, T. (2008). Biological function of fucosylation in
cancer biology. Journal of Biochemistry, 143(6), 725–729. http://dx.doi.org/10.1093/jb/
mvn011.
Mondal, G., Chatterjee, U., Chawla, Y. K., & Chatterjee, B. P. (2011). Alterations of glycan
branching and differential expression of sialic acid on alpha fetoprotein among hepatitis
patients. Glycoconjugate Journal, 28(1), 1–9. http://dx.doi.org/10.1007/s10719-010-
9316-z.
Myers, S. A., Daou, S., Affar, E. B., & Burlingame, A. (2013). Electron transfer dissociation
(ETD): The mass spectrometric breakthrough essential for O-GlcNAc protein
site assignments-a study of the O-GlcNAcylated protein Host Cell Factor C1.
Proteomics, 13(6), 982–991. http://dx.doi.org/10.1002/pmic.201200332 (R. Zahedi &
A. Sickmann, Eds.).
Nakada, H., Numata, Y., Inoue, M., Tanaka, N., Kitagawa, H., Funakoshi, I., et al. (1991).
Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) mono-
clonal antibody (MLS 128). The Journal of Biological Chemistry, 266(19), 12402–12405.
Nanni, P., Panse, C., Gehrig, P., Mueller, S., Grossmann, J., & Schlapbach, R. (2013). PTM
MarkerFinder, a software tool to detect and validate spectra from peptides carrying post-
translational modifications. Proteomics, 13(15), 2251–2255. http://dx.doi.org/10.1002/
pmic.201300036.
Nie, H., Li, Y., & Sun, X.-L. (2012). Recent advances in sialic acid-focused glycomics. Jour-
nal of Proteomics, 75(11), 3098–3112. http://dx.doi.org/10.1016/j.jprot.2012.03.050.
Nie, S., Lo, A., Zhu, J., Wu, J., Ruffin, M. T., & Lubman, D. M. (2013). Isobaric protein-
level labeling strategy for serum glycoprotein quantification analysis by liquid chroma-
tography–tandem mass spectrometry. Analytical Chemistry, 85(11), 5353–5357. http://
dx.doi.org/10.1021/ac400838s.
Nilsson, J., & Larson, G. (2013). Sialic acid capture-and-release and LC-MS(n) analysis of
glycopeptides. Methods in Molecular Biology (Clifton, NJ), 951, 79–100. http://dx.doi.
org/10.1007/978-1-62703-146-2_7.
Nilsson, J., Rüetschi, U., Halim, A., Hesse, C., Carlsohn, E., Brinkmalm, G., et al. (2009).
Enrichment of glycopeptides for glycan structure and attachment site identification.
Nature Methods, 6(11), 809–811. http://dx.doi.org/10.1038/nmeth.1392.
Nishikaze, T., Kawabata, S.-I., Iwamoto, S., & Tanaka, K. (2013). Reversible hydrazide
chemistry-based enrichment for O-GlcNAc-modified peptides and glycopeptides hav-
ing non-reducing GlcNAc residues. The Analyst, 138(23), 7224–7232. http://dx.doi.
org/10.1039/c3an00880k.
Recent Advances in Glycoproteomics 117
Nwosu, C. C., Seipert, R. R., Strum, J. S., Hua, S. S., An, H. J., Zivkovic, A. M., et al.
(2011). Simultaneous and extensive site-specific N- and O-glycosylation analysis in pro-
tein mixtures. Journal of Proteome Research, 10(5), 2612–2624. http://dx.doi.org/10.1021/
pr2001429.
Nyalwidhe, J. O., Betesh, L. R., Powers, T. W., Jones, E. E., White, K. Y., Burch, T. C., et al.
(2013). Increased bisecting N-acetylglucosamine and decreased branched chain glycans
of N-linked glycoproteins in expressed prostatic secretions associated with prostate cancer
progression. Proteomics. Clinical Applications, 7, 677–689. http://dx.doi.org/10.1002/
prca.201200134.
Ongay, S., Boichenko, A., Govorukhina, N., & Bischoff, R. (2012). Glycopeptide enrich-
ment and separation for protein glycosylation analysis. Journal of Separation Science, 35(18),
2341–2372. http://dx.doi.org/10.1002/jssc.201200434.
Orlando, R. (2013). Quantitative analysis of glycoprotein glycans. Methods in Molecular Biol-
ogy (Clifton, NJ), 951, 197–215. http://dx.doi.org/10.1007/978-1-62703-146-2_13.
Otto, M., Wiltfang, J., Cepek, L., Neumann, M., Mollenhauer, B., Steinacker, P., et al.
(2002). Tau protein and 14-3-3 protein in the differential diagnosis of Creutzfeldt-Jakob
disease. Neurology, 58(2), 192–197. http://dx.doi.org/10.1212/wnl.58.2.192.
Otvos, L., Urge, L., & Thurin, J. (1992). Influence of different N- and O-linked carbohy-
drates on the retention times of synthetic peptides in reversed-phase high-performance
liquid chromatography. Journal of Chromatography, 599(1–2), 43–49.
Owen, J. B., Di Domenico, F., Sultana, R., Perluigi, M., Cini, C., Pierce, W. M., et al.
(2009). Proteomics-determined differences in the concanavalin-a-fractionated proteome
of hippocampus and inferior parietal lobule in subjects with Alzheimer’s disease and mild
cognitive Impairment: Implications for progression of AD. Journal of Proteome Research,
8(2), 471–482. http://dx.doi.org/10.1021/pr800667a.
Palmisano, G., Lendal, S. E., Engholm-Keller, K., Leth-Larsen, R., Parker, B. L., &
Larsen, M. R. (2010). Selective enrichment of sialic acid-containing glycopeptides using
titanium dioxide chromatography with analysis by HILIC and mass spectrometry. Nature
Protocols, 5(12), 1974–1982. http://dx.doi.org/10.1038/nprot.2010.167.
Palmisano, G., Melo-Braga, M. N., Engholm-Keller, K., Parker, B. L., & Larsen, M. R.
(2012). Chemical deamidation: A common pitfall in large-scale N-linked glyco-
proteomic mass spectrometry-based analyses. Journal of Proteome Research, 11(3),
1949–1957. http://dx.doi.org/10.1021/pr2011268.
Pan, M., Sun, Y., Zheng, J., & Yang, W. (2013). Boronic acid-functionalized core-shell-shell
magnetic composite microspheres for the selective enrichment of glycoprotein. ACS
Applied Materials & Interfaces, 5(17), 8351–8358. http://dx.doi.org/10.1021/am401285x.
Parker, B. L., Thaysen-Andersen, M., Solis, N., Scott, N. E., Larsen, M. R., Graham, M. E.,
et al. (2013). Site-specific glycan-peptide analysis for determination of N-glycoproteome
heterogeneity. Journal of Proteome Research, 12(12), 5791–5800. http://dx.doi.org/
10.1021/pr400783j.
Percy, A. J., Parker, C. E., & Borchers, C. H. (2013). Pre-analytical and analytical variability
in absolute quantitative MRM-based plasma proteomic studies. Bioanalysis, 5(22),
2837–2856. http://dx.doi.org/10.4155/bio.13.245.
Pernemalm, M., Lewensohn, R., & Lehti€ o, J. (2009). Affinity prefractionation for MS-based
plasma proteomics. Proteomics, 9(6), 1420–1427. http://dx.doi.org/10.1002/
pmic.200800377.
Plasencia, M. D., Isailovic, D., Merenbloom, S. I., Mechref, Y., & Clemmer, D. E. (2008).
Resolving and assigning N-linked glycan structural isomers from ovalbumin by IMS-
MS. Journal of the American Society for Mass Spectrometry, 19(11), 1706–1715. http://dx.
doi.org/10.1016/j.jasms.2008.07.020.
Plavina, T., Wakshull, E., Hancock, W. S., & Hincapie, M. (2007). Combination of abun-
dant protein depletion and multi-lectin affinity chromatography (M-LAC) for plasma
118 Dustin C. Frost and Lingjun Li
hepatocellular carcinoma from liver cirrhosis patients. Acta Biochimica et Biophysica Sinica,
44(9), 765–773. http://dx.doi.org/10.1093/abbs/gms055.
Taga, Y., Kusubata, M., Ogawa-Goto, K., & Hattori, S. (2013). Site-specific quantitative
analysis of overglycosylation of collagen in osteogenesis imperfecta using hydrazide
chemistry and SILAC. Journal of Proteome Research, 12(5), 2225–2232. http://dx.doi.
org/10.1021/pr400079d.
Takátsy, A., B€
oddi, K., Nagy, L., Nagy, G., Szabó, S., Markó, L., et al. (2009). Enrichment of
Amadori products derived from the nonenzymatic glycation of proteins using microscale
boronate affinity chromatography. Analytical Biochemistry, 393(1), 8–22. http://dx.doi.
org/10.1016/j.ab.2009.06.007.
Takegawa, Y., Deguchi, K., Ito, H., Keira, T., Nakagawa, H., & Nishimura, S.-I. (2006).
Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydro-
philic interaction chromatography. Journal of Separation Science, 29(16), 2533–2540.
http://dx.doi.org/10.1002/jssc.200600133.
Tan, H. T., Lee, Y. H., & Chung, M. C. M. (2012). Cancer proteomics. Mass Spectrometry
Reviews, 31(5), 583–605. http://dx.doi.org/10.1002/mas.20356.
Tang, J., Liu, Y., Qi, D., Yao, G., Deng, C., & Zhang, X. (2009). On-plate-selective enrich-
ment of glycopeptides using boronic acid-modified gold nanoparticles for direct
MALDI-QIT-TOF MS analysis. Proteomics, 9(22), 5046–5055. http://dx.doi.org/
10.1002/pmic.200900033.
Tang, J., Liu, Y., Yin, P., Yao, G., Yan, G., Deng, C., et al. (2010). Concanavalin
A-immobilized magnetic nanoparticles for selective enrichment of glycoproteins and
application to glycoproteomics in hepatocelluar carcinoma cell line. Proteomics,
10(10), 2000–2014. http://dx.doi.org/10.1002/pmic.200900377.
Temporini, C., Perani, E., Calleri, E., Dolcini, L., Lubda, D., Caccialanza, G., et al. (2007).
Pronase-immobilized enzyme Reactor: An approach for automation in glycoprotein
analysis by LC/LCESI/MS n. Analytical Chemistry, 79(1), 355–363. http://dx.doi.
org/10.1021/ac0611519.
Teo, C. F., Ingale, S., Wolfert, M. A., Elsayed, G. A., N€ ot, L. G., Chatham, J. C., et al.
(2010). Glycopeptide-specific monoclonal antibodies suggest new roles for
O-GlcNAc. Nature Chemical Biology, 6(5), 338–343. http://dx.doi.org/10.1038/
nchembio.338.
Tep, S., Hincapie, M., & Hancock, W. S. (2012). A general approach for the purification and
quantitative glycomic analysis of human plasma. Analytical and Bioanalytical Chemistry,
402(9), 2687–2700. http://dx.doi.org/10.1007/s00216-012-5712-5.
Thompson, A., Schäfer, J., Kuhn, K., Kienle, S., Schwarz, J., Schmidt, G., et al. (2003). Tan-
dem mass tags: A novel quantification strategy for comparative analysis of complex pro-
tein mixtures by MS/MS. Analytical Chemistry, 75(8), 1895–1904. http://dx.doi.org/
10.1021/ac0262560.
Ueda, K. (2013). Glycoproteomic strategies: From discovery to clinical application of cancer
carbohydrate biomarkers. Proteomics. Clinical Applications. http://dx.doi.org/10.1002/
prca.201200123.
Varki, A. (1993). Biological roles of oligosaccharides: All of the theories are correct.
Glycobiology, 3(2), 97–130.
Walker, S. H., Budhathoki-Uprety, J., Novak, B. M., & Muddiman, D. C. (2011). Stable-
isotope labeled hydrophobic hydrazide reagents for the relative quantification of
N-linked glycans by electrospray ionization mass spectrometry. Analytical Chemistry,
83(17), 6738–6745. http://dx.doi.org/10.1021/ac201376q.
Walker, S. H., Carlisle, B. C., & Muddiman, D. C. (2012). Systematic comparison of reverse
phase and hydrophilic interaction liquid chromatography platforms for the analysis
of N-linked glycans. Analytical Chemistry, 84(19), 8198–8206. http://dx.doi.org/
10.1021/ac3012494.
Recent Advances in Glycoproteomics 121
Walker, S. H., Taylor, A. D., & Muddiman, D. C. (2013). Individuality normalization when
labeling with isotopic glycan hydrazide tags (INLIGHT): A novel glycan-relative quan-
tification strategy. Journal of the American Society for Mass Spectrometry, 24(9), 1376–1384.
http://dx.doi.org/10.1007/s13361-013-0681-2.
Wang, L., Aryal, U. K., Dai, Z., Mason, A. C., Monroe, M. E., Tian, Z.-X., et al. (2012).
Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus
niger using hydrazide chemistry and mass spectrometry. Journal of Proteome Research,
11(1), 143–156. http://dx.doi.org/10.1021/pr200916k.
Wang, C., Fan, W., Zhang, P., Wang, Z., & Huang, L. (2011). One-pot nonreductive
O-glycan release and labeling with 1-phenyl-3-methyl-5-pyrazolone followed by ESI-
MS analysis. Proteomics, 11(21), 4229–4242. http://dx.doi.org/10.1002/pmic.201000677.
Wang, Z., Pandey, A., & Hart, G. W. (2007). Dynamic interplay between O-linked
N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation.
Molecular & Cellular Proteomics, 6(8), 1365–1379. http://dx.doi.org/10.1074/mcp.
M600453-MCP200.
Wang, Z., Udeshi, N. D., O’Malley, M., Shabanowitz, J., Hunt, D. F., & Hart, G. W.
(2010). Enrichment and site mapping of O-linked N-acetylglucosamine by a combina-
tion of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dis-
sociation mass spectrometry. Molecular & Cellular Proteomics, 9(1), 153–160. http://dx.doi.
org/10.1074/mcp.M900268-MCP200.
Wang, Y., Wu, S.-L., & Hancock, W. S. (2006). Approaches to the study of N-linked gly-
coproteins in human plasma using lectin affinity chromatography and nano-HPLC
coupled to electrospray linear ion trap—Fourier transform mass spectrometry.
Glycobiology, 16(6), 514–523. http://dx.doi.org/10.1093/glycob/cwj091.
Wang, C., Wu, Z., Yuan, J., Wang, B., Zhang, P., Zhang, Y., et al. (2013). Simplified quan-
titative glycomics using the stable isotope label Girard’s reagent P by electrospray ioni-
zation mass spectrometry. Journal of Proteome Research, 13(2), 372–384. http://dx.doi.org/
10.1021/pr4010647.
Wei, X., Dulberger, C., & Li, L. (2010). Characterization of murine brain membrane gly-
coproteins by detergent assisted lectin affinity chromatography. Analytical Chemistry,
82(15), 6329–6333.
Wei, X., Herbst, A., Ma, D., Aiken, J., & Li, L. (2011). A quantitative proteomic approach to
prion disease biomarker research: Delving into the glycoproteome. Journal of Proteome
Research, 10(6), 2687–2702. http://dx.doi.org/10.1021/pr2000495.
Wolff, J. J., Leach, F. E., Laremore, T. N., Kaplan, D. A., Easterling, M. L., Linhardt, R. J.,
et al. (2010). Negative electron transfer dissociation of glycosaminoglycans. Analytical
Chemistry, 82(9), 3460–3466. http://dx.doi.org/10.1021/ac100554a.
Woodin, C. L., Maxon, M., & Desaire, H. (2013). Software for automated interpretation of
mass spectrometry data from glycans and glycopeptides. The Analyst, 138(10),
2793–2803. http://dx.doi.org/10.1039/c2an36042j.
Wuhrer, M. (2012). Glycomics using mass spectrometry. Glycoconjugate Journal, 30(1), 11–22.
http://dx.doi.org/10.1007/s10719-012-9376-3.
Wuhrer, M., Catalina, M. I., Deelder, A. M., & Hokke, C. H. (2007). Glycoproteomics
based on tandem mass spectrometry of glycopeptides. Journal of Chromatography. B, Ana-
lytical Technologies in the Biomedical and Life Sciences, 849(1–2), 115–128. http://dx.doi.
org/10.1016/j.jchromb.2006.09.041.
Xu, Y., Wu, Z., Zhang, L., Lu, H., Yang, P., Webley, P. A., et al. (2009). Highly specific
enrichment of glycopeptides using boronic acid-functionalized mesoporous silica. Ana-
lytical Chemistry, 81(1), 503–508. http://dx.doi.org/10.1021/ac801912t.
Xu, Y., Zhang, L., Lu, H., & Yang, P. (2010). On-plate enrichment of glycopeptides by
using boronic acid functionalized gold-coated Si wafer. Proteomics, 10, 1079–1086.
http://dx.doi.org/10.1002/pmic.200900097.
122 Dustin C. Frost and Lingjun Li
Yang, G., Cui, T., Wang, Y., Sun, S., Ma, T., Wang, T., et al. (2013). Selective isolation and
analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera.
Proteomics, 13(9), 1481–1498. http://dx.doi.org/10.1002/pmic.201200259.
Yang, Z., & Hancock, W. S. (2004). Approach to the comprehensive analysis of glycopro-
teins isolated from human serum using a multi-lectin affinity column. Journal of Chroma-
tography. A, 1053(1–2), 79–88. http://dx.doi.org/10.1016/j.chroma.2004.08.150.
Yang, Z., & Hancock, W. S. (2005). Monitoring glycosylation pattern changes of glycopro-
teins using multi-lectin affinity chromatography. Journal of Chromatography. A,
1070(1–2), 57–64.
Yang, Z., Hancock, W. S., Chew, T. R., & Bonilla, L. (2005). A study of glycoproteins in
human serum and plasma reference standards (HUPO) using multilectin affinity chroma-
tography coupled with RPLC-MS/MS. Proteomics, 5(13), 3353–3366. http://dx.doi.
org/10.1002/pmic.200401190.
Yang, Z., Harris, L. E., Palmer-Toy, D. E., & Hancock, W. S. (2006). Multilectin affinity
chromatography for characterization of multiple glycoprotein biomarker candidates in
serum from breast cancer patients. Clinical Chemistry, 52(10), 1897–1905. http://dx.
doi.org/10.1373/clinchem.2005.065862.
Yang, S., & Zhang, H. (2012). Solid-phase glycan isolation for glycomics analysis. Proteomics.
Clinical Applications, 6(11–12), 596–608. http://dx.doi.org/10.1002/prca.201200045.
Ye, H., Boyne, M. T., II, Buhse, L. F., & Hill, J. (2013). Direct approach for qualitative and
quantitative characterization of glycoproteins using tandem mass tags and an LTQ
Orbitrap XL electron transfer dissociation hybrid mass spectrometer. Analytical Chemis-
try, 85(3), 1531–1539. http://dx.doi.org/10.1021/ac3026465.
Yin, X., Bern, M., Xing, Q., Ho, J., Viner, R., & Mayr, M. (2013). Glycoproteomic analysis
of the secretome of human endothelial cells. Molecular & Cellular Proteomics, 12(4),
956–978. http://dx.doi.org/10.1074/mcp.M112.024018.
Yu, X., Huang, Y., Lin, C., & Costello, C. E. (2012). Energy-dependent electron activated
dissociation of metal-adducted permethylated oligosaccharides. Analytical Chemistry,
84(17), 7487–7494. http://dx.doi.org/10.1021/ac301589z.
Yu, X., Jiang, Y., Chen, Y., Huang, Y., Costello, C. E., & Lin, C. (2013). Detailed glycan
structural characterization by electronic excitation dissociation. Analytical Chemistry,
85(21), 10017–10021. http://dx.doi.org/10.1021/ac402886q.
Yue, T., & Haab, B. B. (2009). Microarrays in glycoproteomics research. Clinics in Laboratory
Medicine, 29(1), 15–29. http://dx.doi.org/10.1016/j.cll.2009.01.001.
Yuzwa, S. A., Shan, X., Macauley, M. S., Clark, T., Skorobogatko, Y., Vosseller, K., et al.
(2012). Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggre-
gation. Nature Chemical Biology, 8(4), 393–399. http://dx.doi.org/10.1038/
nchembio.797.
Zaia, J. (2010). Mass spectrometry and glycomics. OMICS: A Journal of Integrative Biology,
14(4), 401–418. http://dx.doi.org/10.1089/omi.2009.0146.
Zauner, G., Deelder, A. M., & Wuhrer, M. (2011). Recent advances in hydrophilic inter-
action liquid chromatography (HILIC) for structural glycomics. Electrophoresis, 32(24),
3456–3466. http://dx.doi.org/10.1002/elps.201100247.
Zauner, G., Koeleman, C. A. M., Deelder, A. M., & Wuhrer, M. (2010). Protein glycosylation
analysis by HILIC-LC-MS of proteinase K-generated N- and O-glycopeptides. Journal
of Separation Science, 33(6–7), 903–910. http://dx.doi.org/10.1002/jssc.200900850.
Zauner, G., Koeleman, C. A. M., Deelder, A. M., & Wuhrer, M. (2012). Mass spectrometric
O-glycan analysis after combined O-glycan release by beta-elimination and
1-phenyl-3-methyl-5-pyrazolone labeling. Biochimica et Biophysica Acta, 1820(9),
1420–1428. http://dx.doi.org/10.1016/j.bbagen.2011.07.004.
Zauner, G., Kozak, R. P., Gardner, R. A., Fernandes, D. L., Deelder, A. M., & Wuhrer, M.
(2012). Protein O-glycosylation analysis. Biological Chemistry, 393(8), 687–708. http://
dx.doi.org/10.1515/hsz-2012-0144.
Recent Advances in Glycoproteomics 123
Zeng, Z., Hincapie, M., Pitteri, S. J., Hanash, S., Schalkwijk, J., Hogan, J. M., et al. (2011).
A proteomics platform combining depletion, multi-lectin affinity chromatography (M-
LAC), and isoelectric focusing to study the breast cancer proteome. Analytical Chemistry,
83(12), 4845–4854. http://dx.doi.org/10.1021/ac2002802.
Zhang, H., Li, X.-J., Martin, D. B., & Aebersold, R. (2003). Identification and quantification
of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass
spectrometry. Nature Biotechnology, 21(6), 660–666. http://dx.doi.org/10.1038/nbt827.
Zhang, L., Lu, H., & Yang, P. (2009). Specific enrichment methods for glycoproteome
research. Analytical and Bioanalytical Chemistry, 396(1), 199–203. http://dx.doi.org/
10.1007/s00216-009-3086-0.
Zhang, Q., Schepmoes, A. A., Brock, J. W. C., Wu, S., Moore, R. J., Purvine, S. O., et al.
(2008). Improved methods for the enrichment and analysis of glycated peptides. Analyt-
ical Chemistry, 80(24), 9822–9829. http://dx.doi.org/10.1021/ac801704j.
Zhang, B., Sheng, Q., Li, X., Liang, Q., Yan, J., & Liang, X. (2011). Selective enrichment of
glycopeptides for mass spectrometry analysis using C18 fractionation and titanium diox-
ide chromatography. Journal of Separation Science, 34(19), 2745–2750. http://dx.doi.org/
10.1002/jssc.201100427.
Zhang, Q., Tang, N., Brock, J. W. C., Mottaz, H. M., Ames, J. M., Baynes, J. W., et al.
(2007). Enrichment and analysis of nonenzymatically glycated peptides: Boronate affinity
chromatography coupled with electron-transfer dissociation mass spectrometry. Journal of
Proteome Research, 6(6), 2323–2330. http://dx.doi.org/10.1021/pr070112q.
Zhang, W., Wang, H., Zhang, L., Yao, J., & Yang, P. (2011). Large-scale assignment of
N-glycosylation sites using complementary enzymatic deglycosylation. Talanta, 85(1),
499–505. http://dx.doi.org/10.1016/j.talanta.2011.04.019.
Zhang, L., Xu, Y., Yao, H., Xie, L., Yao, J., Lu, H., et al. (2009). Boronic acid functionalized
core-satellite composite nanoparticles for advanced enrichment of glycopeptides and gly-
coproteins. Chemistry (Weinheim an der Bergstrasse, Germany), 15(39), 10158–10166.
http://dx.doi.org/10.1002/chem.200901347.
Zhao, Y., Jia, W., Wang, J., Ying, W., Zhang, Y., & Qian, X. (2011). Fragmentation and
site-specific quantification of core fucosylated glycoprotein by multiple reaction
monitoring-mass spectrometry. Analytical Chemistry, 83(22), 8802–8809. http://dx.
doi.org/10.1021/ac201676a.
Zhou, Y., Aebersold, R., & Zhang, H. (2007). Isolation of N-linked glycopeptides from
plasma. Analytical Chemistry, 79(15), 5826–5837. http://dx.doi.org/10.1021/ac0623181.
Zhou, W., Yao, N., Yao, G., Deng, C., Zhang, X., & Yang, P. (2008). Facile synthesis of
aminophenylboronic acid-functionalized magnetic nanoparticles for selective separation
of glycopeptides and glycoproteins. Chemical Communications (Cambridge, England), (43),
5577–5579. http://dx.doi.org/10.1039/b808800d.
Zhu, J., He, J., Liu, Y., Simeone, D. M., & Lubman, D. M. (2012). Identification of glyco-
protein markers for pancreatic cancer CD24+CD44 + stem-like cells using nano-LC-
MS/MS and tissue microarray. Journal of Proteome Research, 11(4), 2272–2281. http://
dx.doi.org/10.1021/pr201059g.
Zhu, Z., Hua, D., Clark, D. F., Go, E. P., & Desaire, H. (2013). GlycoPep Detector: A tool
for assigning mass spectrometry data of N-linked glycopeptides on the basis of their elec-
tron transfer dissociation spectra. Analytical Chemistry, 85(10), 5023–5032. http://dx.doi.
org/10.1021/ac400287n.
Zielinska, D. F., Gnad, F., Wiśniewski, J. R., & Mann, M. (2010). Precision mapping of an
in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell,
141(5), 897–907. http://dx.doi.org/10.1016/j.cell.2010.04.012.
CHAPTER FOUR
Contents
1. Introduction 126
2. An Integrated Proteogenomics Protocol for Personalized Dentistry 126
2.1 Human samples 126
2.2 Bioinformatics analysis 130
2.3 Proteomics technologies, with a focus on the label-free tools 138
3. Oral Diseases 145
3.1 Dental caries 146
3.2 Periodontitis 147
3.3 Oral lichen planus 149
3.4 Oral cancer 151
4. Concluding Remarks 151
References 151
Abstract
Design and implementation of new biocompatible materials and achievements in the
field of nanogenomics and nanoproteomics as well as in other related and allied sci-
ences in the broader framework of translational and clinical nanomedicine are paving
new avenues for nanodentistry. Classical dentistry is becoming more predictive, preven-
tive, personalized, and participatory, providing the patients with a tailored and targeted
treatment and handling of their diseases. Considering the global impact of the oral
pathologies, being particularly heavy in underdeveloped and developing countries, it
is mandatory from an ethical perspective to ensure a global oral health.
Nanobiotechnologies play a major role in this ambitious goal. In this review, we will
focus on the bioinformatics, nanogenomics, and nanoproteomics aspects of contem-
porary nanodentistry, emphasizing the urgent need for an integrated proteogenomics
approach and addressing its clinical and translational implications and new future per-
spectives and scenarios.
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 125
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00004-X
126 Nicola Luigi Bragazzi et al.
1. INTRODUCTION
Advancements in the field of oral biomaterials (Choi, Ben-Nissan,
Matinlinna, & Conway, 2013; Covani et al., 2007; Mallineni, Nuvvula,
Matinlinna, Yiu, & King, 2013; Marconcini et al., 2014; Riley,
Bavastrello, Covani, Barone, & Nicolini, 2005; Zandparsa, 2014), nano-
technologies (Ozak & Ozkan, 2013) and nanobiotechnologies, such as
nanogenomics (Nicolini, 2006, 2010) and nanoproteomics (Kobeissy
et al., 2014; Nicolini & Pechkova, 2010a,2010b) tools as fundamental com-
ponents of a modern nanobiomedical approach (Nicolini et al., 2012;
Nicolini, Bragazzi, & Pechkova, 2012; Nicolini, Bragazzi, & Pechkova,
2013) have enabled the birth of a new, highly interdisciplinary and rapidly
growing discipline, termed as nanodentistry (Freitas, 2000; Kanaparthy &
Kanaparthy, 2011; Mantri & Mantri, 2013), emerging from complementary
and converging approaches.
Early diagnosing and properly monitoring oral diseases, avoiding their
recurrence, providing the patients with a tailored, individualized, and targeted
treatment (Bragazzi, 2013a, 2013b, 2013c) are important tasks within the field
of personalized dentistry (Garcia et al., 2013; Glurich et al., 2013; Kornman &
Duff, 2012; Razzouk & Termechi, 2013), that is becoming more predictive,
preventive and participatory (Cafiero & Matarasso, 2013). Oral diseases have a
tremendous burden and societal impact, affecting approximately 3.9 billions of
people worldwide and particularly in underdeveloped and developing coun-
tries (Richards, 2013), and therefore ensuring global oral health is an ethical
onus (Giannobile, 2013).
In this review, we will focus on the bioinformatics, nanogenomics, and
nanoproteomics aspects of contemporary nanodentistry, addressing its clin-
ical and translational implications and foreseeing its new future perspectives
and scenarios.
field of oral dentistry, each one with its own peculiarity and advantages, as
well as pitfalls and drawback (Fig. 4.1). Once obtained, data can be eventu-
ally combined in order to have a robust molecular signature and a panel of
selected, differentially expressed markers, that need to be replicated and val-
idated before entering everyday clinical practice and routine. A bio-marker
is indeed defined as reliable, reproducible, sensitive, and specific (Strimbu &
Tavel, 2010). In the following subsections, we briefly overview the main
sources of biomarkers in the field of oral pathologies, namely tissues biopsies,
blood, dental plaque and oral biofilms, gingival crevicular fluid (GCF),
saliva, and oral rinse.
2.1.1 Tissues
Oral cavity is a multifunctional environment made up of different compo-
nents, building up a complex architecture. Its anatomy includes tissues from
the mucosa (which is divided into different parts, namely the labial, buccal,
2.1.2 Blood
Blood is a bodily fluid that delivers nutrients, oxygen, and other fundamental
molecules for life, clearing and removing cellular waste products. It is a very
common and popular, accepted sample, which is easy-to-obtain, and not
difficult to store and process. It can be used as a whole blood or selected puri-
fied components.
In oral diseases, blood-derived biomarkers are associated with systemic
risks and pathologies, such as cardiovascular (Meurman, Janket,
Qvarnstr€ om, & Nuutinen, 2003), rheumatological ( Joseph, Rajappan,
Nath, & Paul, 2013; Kobayashi et al., 2014; Okada et al., 2013), gastroin-
testinal ( Jaiswal, Deo, Bhongade, & Jaiswal, 2011), and metabolic
(Pradeep, Kumari, Kalra, & Priyanka, 2013) diseases. Some studies have cor-
related blood with other samples like saliva (Haririan et al., 2012; Sundar,
Krishnan, Krishnaraj, Hemalatha, & Alam, 2013), GCF (Fiorini et al.,
2013; Gokul, Faizuddin, & Pradeep, 2012; Patel & Raju, 2013; Pradeep
et al., 2011; Raghavendra et al., 2012; Sharma, Pradeep, Raghavendra,
Arjun, & Kathariya, 2012; Thorat, Pradeep, & Garg, 2010), finding a pos-
itive correlation, even though in few cases not always concordant (Fiorini
et al., 2013).
2.1.5 Saliva
Saliva is a complex biological fluid (Huang, 2004; Ruhl, 2012; Wong, 2009;
Ogawa et al., 2011; Zhang et al., 2013), produced by major salivary glands
(submandibular, sublingual and parotid glands) and minor ones (scattered
throughout the entire oral mucosa), and made up of water for the
99–99.5% (594–1194 mL/day) and of a mixture of microorganisms (bacte-
ria, viruses, fungi, protozoa), (Jagtap et al., 2012; Wong, 2009) ions,
enzymes and catalytic proteins, DNA and RNA, hormones, desquamated
cells, food debris, and other molecules for the remaining 0.5–1%
(3–12 mL/day), ranging up to 4–5% ore more (24–60 mL/day) in some
clinical cases (Wong, 2009). Moreover, being the oral cavity in intimate
contact with the gastrointestinal and respiratory tracts (Wong, 2009), it
may contain also expectorated bronchial and nasal secretions, typical gastro-
intestinal or respiratory microorganisms, and some serum constituents that
are derived from the local vasculature of the salivary glands and GCF, as well
as from oral wounds (Deepa & Thirrunavukkarasu, 2010). Its production is
finely tuned by the autonomic system, at least for the exocrine components
(Wong, 2009), and plays a role in different functions, from speech and pho-
nation, bolus formation and swallowing, starch digestion, protection, lubri-
cation, buffering action, maintenance of tooth integrity through
maintenance of an adequate level of mineralization, to perception of taste.
Its proteome has unique features that makes it different from other
130 Nicola Luigi Bragazzi et al.
Figure 4.2 The algorithm on which the leader-gene tool for molecular genomics is
based and a screen-shot of the software.
138 Nicola Luigi Bragazzi et al.
The clustering techniques the user can choose are Clustering K-means
and Chinese whispers (which has been thought specifically for graph clus-
tering); as far as the number of clusters is concerned, the user can choose
from heuristic number or provided by the user himself.
The obtained list of Class A and Class B genes can be used for predicting
further biomarkers such as miRNAs (work currently in progress) or being
validated with ad hoc experiments, such as gene microarrays or protein arrays
after being expressed and subsequently analyzed via labeled or label-free
nanobiotechnologies (Fig. 4.1), which are better described in the following
paragraphs.
Nicolini, Bragazzi, and Pechkova (2012), Nicolini et al. (2013), Nicolini et al.
(2013), and Spera et al. (2010), their experimental mass lists with that of the
known samples (P53, JUN, CdK2, CdKN1A).
We can then conservatively conclude that the implemented chemistry and
analysis for the first time demonstrate the successful use of MS for the
characterization of proteins immobilized on NAPPA. Further development
is in progress to bring this label-free procedure to practice as an adjunct to
fluorescence NAPPA work, which has already seen significant clinical appli-
cations in the last decades (Anderson et al., 2008; Nand, Gautam, Pérez,
Merino, & Zhu, 2012; Nicolini & Pechkova, 2010a,2010b; Sibani &
LaBaer, 2011; Spera et al., 2013b). NAPPA approach has been indeed used
for investigating cancer (Anderson et al., 2011, 2010), type 1 diabetes
(Miersch et al., 2013), rheumatological diseases (Gibson et al., 2012;
Wright et al., 2012), and infections (Ceroni et al., 2010; Manzano-Román
et al., 2012; Montor et al., 2009; Rolfs et al., 2008; Thanawastien,
Montor, Labaer, Mekalanos, & Yoon, 2009).
The background generated by the reticulocytes lysate is, however, still
significant and need to be reduced to make this approach routinely applica-
ble in the clinics. This reduction might be instead achieved by the use of a
bacterial cell-free expression system with respect to the traditional mamma-
lian lysate, particularly required by the highly sensitive nanotechnologies
being here utilized. The application of bacterial PURExpress to NAPPA
(in progress) consists of a template double-stranded DNA containing the
gene of interest fused to a SNAP tag and the upstream T7 promoter
(Nicolini, Spera et al., 2013; Pechkova et al., 2010).
By adding the PURExpress reconstituted cell-free translation system
(Houlihan, Gatti-Lafranconi, Kaltenbach, Lowe, & Hollfelder, 2014), the
template DNA is transcribed into mRNA, and then translated into a fusion
protein containing the N-terminal SNAP tag and the C-terminal target pro-
tein. In the same spot, the SNAP tag allows the synthesized protein to bind
to its own template DNA via the BG linkage, thus immobilizes the target
protein. The rest of the reaction mixture can be washed away and the
immobilized target protein is allowed to interact with a mixture of query
proteins. After the binding reaction, the unbound proteins are washed away
and the target protein complex is released by cleaving the template DNA.
To compare the backgrounds of NAPPA between PURExpress (bacte-
rial lysate) and RRL (rabbit reticulocyte lysate) by Mass Spectrometry (MS)
and Fluorescence we are presently utilizing NEB in vitro system and SNAP
fusion as an alternative to in vitro system and GST tag.
Proteomics and Proteogenomics Approaches for Oral Diseases 141
Their advantages are higher expression level and cleaner for downstream
analysis, making possible and really effective Label Free quantitative analysis
at the nanoscale (work in progress in cooperation with Arizona State Uni-
versity, ASU, and New England Biolabs, NEB; Nicolini et al., 2013;
Nicolini et al., 2013).
(Reddy et al., 2011), but we focused on NAPPA since it offers many advan-
tages in comparison with classical technologies.
Recently, we have developed a new device that couples NAPPA with the
quartz crystal microbalance with dissipation factor monitoring (QCM_D;
Nicolini, Adami, et al., 2012; Nicolini, Bragazzi, & Pechkova, 2012; Spera
et al., 2013b).
The QCM_D instrument was developed by Elbatech Srl. The quartz
was connected to an RF gain-phase detector (Analog Devices, Inc.,
Norwood, MA, USA) and was driven by a precision DDS (Analog Devices,
Inc., Norwood, MA, USA) around its resonance frequency, thus acquiring
a conductance versus frequency curve (conductance curve) which shows
a typical Gaussian behavior. The conductance curve peak was at the
actual resonance frequency while the shape of the curve indicated how
the viscoelastic effects of the surrounding layers affected the oscillation.
The QCM_D-dedicated software, QCMAgic-Q5.3.256 (Elbatech srl,
Marciana—LI, Italy) allows to acquire the conductance curve or the fre-
quency and dissipation factor variation versus time. In order to have a stable
control of the temperature, the experiments were conducted in a tempera-
ture chamber. Microarrays were produced on standard nanogravimetry qua-
rtz used as highly sensitive transducers. The QC expressing proteins
consisted of 9.5 MHz, AT-cut quartz crystal of 14 mm blank diameter
and 7.5 mm electrode diameter, produced by ICM (Oklahoma City,
OK, USA). The electrode material was 100 Å Cr and 1000 Å Au and the
quartz was embedded into glass-like structures for easy handling.
The NAPPA-QC arrays were printed with 100 spots per QC.
Quartzes gold surfaces were coated with cysteamine to allow the immo-
bilization of the NAPPA printing mix. Briefly, quartzes were washed 3
with ethanol, dried with Argon and incubated over night at 4 C with
2 mM cysteamine. Quartzes were then washed 3 with ethanol to remove
any unbound cysteamine and dried with Argon. Plasmids DNA coding for
GST tagged proteins were transformed into E. coli and DNA were purified
using the NucleoPrepII anion exchange resin (Macherey Nagel). NAPPA
printing mix was prepared with 1.4 mg/ml DNA, 3.75 mg/ml BSA
(Sigma–Aldrich), 5 mM BS3 (Pierce, Rockford, IL, USA), and 66.5 mg
polyclonal capture GST antibody (GE Healthcares). Negative controls,
named master mix (hereinafter abbreviated as “MM”), were obtained
replacing DNA for water in the printing mix. Samples were incubated at
room temperature for 1 h with agitation and then printed on the
cysteamine-coated gold quartz using the Qarray II from Genetix. In order
144 Nicola Luigi Bragazzi et al.
to enhance the sensitivity, each quartz was printed with 100 identical fea-
tures of 300 m diameter each, spaced by 350 m center-to-center.
Gene expression was performed immediately before the assay, following
the protocol described in Spera et al. (2013b). Briefly, in vitro transcription and
translation (IVTT) were performed using HeLa lysate mix (1-Step Human
Coupled IVTT Kit, Thermo Fisher Scientific Inc.), prepared according to
the manufacturers’ instructions. The quartz, connected to the nanogravimeter
inside the incubator, was incubated for 10 min at 30 C with 40 ml of HeLa
lysate mix for proteins synthesis and then, the temperature was decreased to
15 C for a period of 5 min to facilitate the proteins binding on the capture
antibody (anti-GST). After the protein expression and capture, the quartz was
removed from the instrument and washed at room temperature, in 500 mM
NaCl PBS for 3. The protocol described above was followed identically for
both negative control QC (the one with only MM, i.e., all the NAPPA chem-
istry except the cDNA) and protein displaying QC.
After protein expression, capture, and washing the QCs were used for
the interaction studies QC displaying the expressed protein was spotted with
40 ml of the desired molecule solutions in PBS at increasing concentrations
at 22 C.
We also tested the possibility to analyze drug/small molecule–protein
interactions in QC displaying multiple proteins, a task which is not possible
with fluorescence based arrays (Spera et al., 2013a,2013b).
QCM_D measures were calibrated for frequency and for D factor shifts.
The calibration curves equation (obtained with Ordinary Least Squares
methods, OLS) are (Spera et al., 2013a,2013b):
Figure 4.3 Conductance curves for the NAPPA-Quartz Crystal expressing p53, CDK2,
and Jun (all the cDNAs being co-immobilized in the same feature).
The coefficients of variations yield values that are usually very low, con-
firming the repeatability of the experiments and the validity and portability
of the technique. In our hands, NAPPA-based QCM_D proved to have an
intra-assay overall CV of 5% (range 3.3–8.0%; Spera et al., 2013b).
In conclusion, our innovative conductometer, realized by combining
NAPPA technology with QCM_D, enables the study of genes and their
products, the characterization of protein–protein and protein–drugs/small
molecules interactions in a multiparametric way, taking advantage of the
multiple information provided by the analysis of the conductance curves
(i.e., conductance, viscoelasticity, and adsorbed mass). Moreover, through
our conductometer it is possible to acquire detailed information about
the kinetic constants of the interaction.
All these approaches can be combined and together can provide useful
information (Fig. 4.4).
3. ORAL DISEASES
Oral diseases are complex pathologies, deriving from the intersection
of different components: the oral microbial flora (microbiome), environ-
mental and behavioral factors and life styles, the human genetic make-up
146 Nicola Luigi Bragazzi et al.
Figure 4.4 MALDI-TOF spectra of NAPPA after protein trypsin digestion, 5–20 kDa
range, for p53 (upper, left) versus A (bottom, left) samples. p53 Normalized conductance
curve acquired with the NAPPA-QCM_C conductomer (right). Proteomics approaches
can be combined in order to get more information.
(the genome), its transcription and translation (the transcriptome, the pro-
teome, the metabolome, or metabonome and further levels).
For this reason, all the approaches that we have overviewed in the pre-
vious sections should be coherently integrated into a proper framework.
3.2. Periodontitis
Periodontitis is a set of inflammatory diseases affecting the periodontium,
that is, the tissues that surround and support the teeth. Periodontitis involves
progressive loss of the alveolar bone around the teeth, and if left untreated,
can lead to the loosening and subsequent resorption and loss of teeth. Peri-
odontitis is caused by microorganisms that adhere to and grow on the tooth’s
surfaces, along with an overly aggressive immune response against these
microorganisms.
Until 1977, periodontitis was divided into two classes (juvenile and
chronic marginal periodontitis), that have become four in 1986 (the first class
has been split into subclasses, prepubertal, localized and generalized, the
other classes including adult, necrotizing ulcerative gingivo-periodontitis,
148 Nicola Luigi Bragazzi et al.
Figure 4.5 Up- and downregulated genes involved in pathogenesis of OLP. In black:
genes for which there are no or little information about expression; in light grey, neutral
genes in OLP disease; in grey, upregulated genes in OLP disease; in dark grey, down-
regulated genes in OLP disease (top). Plot of disease-related connectivities (WNL,
weighted number of links) versus global connectivities (TIS, total interactions score).
Calculated leader genes are above the regression tendency line (bottom).
4. CONCLUDING REMARKS
Impressive progresses have been made in the last decades. New bio-
informatics tools and resources have been designed, as well genomics, meta-
genomics and proteomics approaches that have a great added clinical value.
Interestingly, integrated proteogenomics approaches have lead to models
which have been proven superior to those including only data deriving from
a single omics technology.
Our bioinformatics algorithm enables the prioritization and selection of
few genes that can be subsequently expressed within the NAPPA array. Our
conductomer appears promising in analyzing multigene and -protein inter-
actions and seems to overcome most difficulties and hurdles of the classical
techniques. Being versatile, it can be used in studying gene–gene, gene–pro-
tein, protein–protein, gene–drug, and protein–drug interactions.
However, some limitations remain, such as those due to the usually small
size of the performed clinical trials and studies (Skates et al., 2013), that hin-
der the power of the investigations themselves and the generalizability of
their findings. Efforts should be undertaken in this direction, in order to pro-
vide reliable results that can be translated into the clinical practice in order to
provide the patients a tailored treatment.
REFERENCES
Ai, J. Y., Smith, B., & Wong, D. T. (2012). Bioinformatics advances in saliva diagnostics.
International Journal of Oral Science, 4(2), 85–87.
Al-Tarawneh, S. K., Border, M. B., Dibble, C. F., & Bencharit, S. (2011). Defining salivary
biomarkers using mass spectrometry-based proteomics: A systematic review. OMICS,
15(6), 353–361.
152 Nicola Luigi Bragazzi et al.
Amado, F. M., Ferreira, R. P., & Vitorino, R. (2013). One decade of salivary
proteomics: Current approaches and outstanding challenges. Clinical Biochemistry,
46(6), 506–517.
Anderson, K. S., Ramachandran, N., Wong, J., Raphael, J. V., Hainsworth, E.,
Demirkan, G., et al. (2008). Application of protein microarrays for multiplexed detection
of antibodies to tumor antigens in breast cancer. Journal of Proteome Research, 7(4),
1490–1499.
Anderson, K. S., Sibani, S., Wallstrom, G., Qiu, J., Mendoza, E. A., Raphael, J., et al. (2011).
Protein microarray signature of autoantibody biomarkers for the early detection of breast
cancer. Journal of Proteome Research, 10(1), 85–96.
Anderson, K. S., Wong, J., Vitonis, A., Crum, C. P., Sluss, P. M., Labaer, J., et al. (2010). p53
autoantibodies as potential detection and prognostic biomarkers in serous ovarian cancer.
Cancer Epidemiology, Biomarkers & Prevention, 19(3), 859–868.
Andreasen, J. O. (1968). Oral lichen planus. 1. A clinical evaluation of 115 cases. Oral Surgery,
Oral Medicine, and Oral Pathology, 25(1), 31–42.
Arrais, J. P., Rosa, N., Melo, J., Coelho, E. D., Amaral, D., Correia, M. J., et al. (2013).
OralCard: A bioinformatic tool for the study of oral proteome. Archives of Oral Biology,
58(7), 762–772.
Bánóczy, J., & Rugg-Gunn, A. (2013). Epidemiology and prevention of dental caries. Acta
Medicine Academica, 42(2), 105–107.
Bassim, C. W., Ambatipudi, K. S., Mays, J. W., Edwards, D. A., Swatkoski, S., Fassil, H.,
et al. (2012). Quantitative salivary proteomic differences in oral chronic graft-versus-host
disease. Journal of Clinical Immunology, 32(6), 1390–1399.
Becker, K. G., Barnes, K. C., Bright, T. J., & Wang, S. A. (2004). The genetic association
database. Nature Genetics, 36(5), 431–432.
Belmonte, L., Spera, R., & Nicolini, C. (2013). SpADS: An R script for mass spectrometry
data preprocessing before data mining. Journal of Computer Science and Systems Biology, 6,
298–304.
Bencharit, S., Altarawneh, S. K., Baxter, S. S., Carlson, J., Ross, G. F., Border, M. B., et al.
(2012). Elucidating role of salivary proteins in denture stomatitis using a proteomic
approach. Molecular BioSystems, 8(12), 3216–3223.
Benson, D. A., Karsch-Mizrachi, I., Clark, K., Lipman, D. J., Ostell, J., & Sayers, E. W.
(2012). GenBank. Nucleic Acids Research, 40, D48–D53 (Database issue).
Bragazzi, N. L. (2013a). Children, adolescents, and young adults participatory medicine:
Involving them in the health care process as a strategy for facing the infertility issue.
The American Journal of Bioethics, 13(3), 43–44.
Bragazzi, N. L. (2013b). From P0 to P6 medicine, a model of highly participatory, narrative,
interactive, and “augmented” medicine: Some considerations on Salvatore Iaconesi’s
clinical story. Patient Preference and Adherence, 7, 353–359.
Bragazzi, N. L. (2013c). Rethinking psychiatry with OMICS science in the age of person-
alized P5 medicine: Ready for psychiatome? Philosophy, Ethics, and Humanities in Medi-
cine, 8(1), 4.
Bragazzi, N., Giacomelli, L., Sivozhelezov, V., & Nicolini, C. (2011). LeaderGene: A fast
data-mining tool for molecular genomics. Journal of Proteomics & Bioinformatics, 4(4),
083–085.
Bragazzi, N. L., & Nicolini, C. (2013). A leader genes approach-based tool for molecular
genomics: From gene-ranking to gene-network systems biology and biotargets predic-
tions. Journal of Computer Science and Systems Biology, 6, 165–176.
Braud, C., Baeten, D., Giral, M., Pallier, A., Ashton-Chess, J., Braudeau, C., et al. (2008).
Immunosuppressive drug-free operational immune tolerancein human kidney transplant
recipients: Part I. Blood gene expression statistical analysis. Journal of Cellular Biochemistry,
103(6), 1681–1692.
Proteomics and Proteogenomics Approaches for Oral Diseases 153
Cafiero, C., & Matarasso, S. (2013). Predictive, preventive, personalised and participatory
periodontology: ‘The 5Ps age’ has already started. The EPMA Journal, 4(1), 16. http://
dx.doi.org/10.1186/1878-5085-4-16.
Carbone, M., Arduino, P. G., Carrozzo, M., Gandolfo, S., Argiolas, M. R., Bertolusso, G.,
et al. (2009). Course of oral lichen planus: A retrospective study of 808 northern Italian
patients. Oral Diseases, 15(3), 235–243.
Ceroni, A., Sibani, S., Baiker, A., Pothineni, V. R., Bailer, S. M., LaBaer, J., et al. (2010).
Systematic analysis of the IgG antibody immune response against varicella zoster virus
(VZV) using a self-assembled protein microarray. Molecular BioSystems, 6(9), 1604–1610.
Chen, T., Abbey, K., Deng, W. J., & Cheng, M. C. (2005). The bioinformatics resource for
oral pathogens. Nucleic Acids Research, 33, W734–W740 (Web Server issue).
Chen, Y. T., Chong, Y. M., Cheng, C. W., Ho, C. L., Tsai, H. W., Kasten, F. H., et al.
(2013). Identification of novel tumor markers for oral squamous cell carcinoma using
glycoproteomic analysis. Clinica Chimica Acta, 420, 45–53.
Chen, T., Yu, W. H., Izard, J., Baranova, O. V., Lakshmanan, A., & Dewhirst, F. E. (2010).
The Human Oral Microbiome Database: A web accessible resource for investigating oral
microbe taxonomic and genomic information. Database (Oxford), 2010, baq013.
Cheng, W. C., Tsai, M. L., Chang, C. W., Huang, C. L., Chen, C. R., Shu, W. Y., et al.
(2010). Microarray meta-analysis database (M(2)DB): A uniformly pre-processed, quality
controlled, and manually curated human clinical microarray database. BMC Bioinformat-
ics, 11, 421.
Choi, A. H., Ben-Nissan, B., Matinlinna, J. P., & Conway, R. C. (2013). Current perspec-
tives: Calcium phosphate nanocoatings and nanocomposite coatings in dentistry. Journal
of Dental Research, 92(10), 853–859.
Covani, U., Giacomelli, L., Krajewski, A., Ravaglioli, A., Spotorno, L., Loria, P., et al.
(2007). Biomaterials for orthopedics: A roughness analysis by atomic force microscopy.
Journal of Biomedical Materials Research. Part A, 82(3), 723–730.
Covani, U., Marconcini, S., Giacomelli, L., Sivozhelevov, V., Barone, A., & Nicolini, C.
(2008 Oct). Bioinformatic prediction of leader genes in human periodontitis. Journal
of Periodontology, 79(10), 1974–1983.
Covani, U., Marconcini, S., Derchi, G., Barone, A., & Giacomelli, L. (2009). Relationship
between human periodontitis and type 2 diabetes at a genomic level: A data-mining
study. Journal of Periodontology, 80(8), 1265–1273.
Cuevas-Córdoba, B., & Santiago-Garcı́a, J. (2014). Saliva: A fluid of study for OMICS.
Omics: A Journal of Integrative Biology, 18(2), 87–97.
Deepa, T., & Thirrunavukkarasu, N. (2010). Saliva as a potential diagnostic tool. Indian Jour-
nal of Medical Sciences, 64(7), 293–306.
De La Iglesia, D., Chiesa, S., Kern, J., Maojo, V., Martin-Sanchez, F., Potamias, G., et al.
(2009). Nanoinformatics: New challenges for biomedical informatics at the nano level.
Studies in Health Technology and Informatics, 150, 987–991.
Dewhirst, F. E., Chen, T., Izard, J., Paster, B. J., Tanner, A. C., Yu, W. H., et al. (2010). The
human oral microbiome. Journal of Bacteriology, 192(19), 5002–5017.
Dimitrov, D. V., & Hoeng, J. (2013). Systems approaches to computational modeling of the
oral microbiome. Frontiers in Physiology, 4, 172.
Doğan, R., Leaman, R., & Lu, Z. (2014). NCBI disease corpus: A resource for disease name
recognition and concept normalization. Journal of Biomedical Informatics, 47, 1–10. http://
dx.doi.org/10.1016/j.jbi.2013.12.006, pii: S1532-0464(13)00197-4.
Duran-Pinedo, A. E., Paster, B., Teles, R., & Frias-Lopez, J. (2011). Correlation network
analysis applied to complex biofilm communities. PLoS One, 6(12), e28438.
Edgar, R., Domrachev, M., & Lash, A. E. (2002). Gene Expression Omnibus: NCBI
gene expression and hybridization array data repository. Nucleic Acids Research, 30(1),
207–210.
154 Nicola Luigi Bragazzi et al.
Franceschini, A., Szklarczyk, D., Frankild, S., Kuhn, M., Simonovic, M., Roth, A., et al.
(2013). STRING v9.1: Protein-protein interaction networks, with increased coverage
and integration. Nucleic Acids Research, 41(Database issue), D808–D815.
Fiorini, T., Susin, C., da Rocha, J. M., Weidlich, P., Vianna, P., Moreira, C. H., et al. (2013).
Effect of nonsurgical periodontal therapy on serum and gingival crevicular fluid
cytokine levels during pregnancy and postpartum. Journal of Periodontal Research, 48(1),
126–133.
Freitas, R. A., Jr. (2000). Nanodentistry. The Journal of the American Dental Association,
131(11), 1559–1565.
Gadewal, N. S., & Zingde, S. M. (2011). Database and interaction network of genes involved
in oral cancer: Version II. Bioinformation, 6(4), 169–170.
Gaiser, S., Deyhle, H., Bunk, O., White, S. N., & Müller, B. (2012). Understanding nano-
anatomy of healthy and carious human teeth: A prerequisite for nanodentistry.
Biointerphases, 7(1–4), 4.
Gandolfo, S., Richiardi, L., Carrozzo, M., Broccoletti, R., Carbone, M., Pagano, M., et al.
(2004). Risk of oral squamous cell carcinoma in 402 patients with oral lichen planus:
A follow-up study in an Italian population. Oral Oncology, 40(1), 77–83.
Garcia, I., Kuska, R., & Somerman, M. J. (2013). Expanding the foundation for personalized
medicine: Implications and challenges for dentistry. Journal of Dental Research, 92(7
Suppl), 3S–10S.
Ghannoum, M. A., Jurevic, R. J., Mukherjee, P. K., Cui, F., Sikaroodi, M., Naqvi, A., et al.
(2010). Characterization of the oral fungal microbiome (mycobiome) in healthy individ-
uals. PLoS Pathogen, 6(1), e1000713.
Giacomelli, L., & Nicolini, C. (2006). Gene expression of human T lymphocytes cell cycle:
Experimental and bioinformatic analysis. Journal of Cellular Biochemistry, 99(5),
1326–1333.
Giacomelli, L., Oluwadara, O., Chiappe, G., Barone, A., Chiappelli, F., & Covani, U.
(2009). Relationship between human oral lichen planus and oral squamous cell carci-
noma at a genomic level: A datamining study. Bioinformation, 4(6), 258–262.
Giacomelli, L., & Covani, U. (2010). Bioinformatics and data mining studies in oral genomics
and proteomics: New trends and challenges. The Open Dentistry Journal, 4, 67–71.
Giannobile, W. V. (2013). Our duty to promote global oral health. Journal of Dental Research,
92(7), 573–574.
Gibson, D. S., Qiu, J., Mendoza, E. A., Barker, K., Rooney, M. E., & LaBaer, J. (2012).
Circulating and synovial antibody profiling of juvenile arthritis patients by nucleic acid
programmable protein arrays. Arthritis Research & Therapy, 14(2), R77.
Glaab, E., Garibaldi, J. M., & Krasnogor, N. (2009). ArrayMining: A modular web-
application for microarray analysis combining ensemble and consensus methods with
cross-study normalization. BMC Bioinformatics, 10, 358.
Glurich, I., Acharya, A., Shukla, S. K., Nycz, G. R., & Brilliant, M. H. (2013).
The oral-systemic personalized medicine model at Marshfield Clinic. Oral Diseases,
19(1), 1–17.
Gokul, K., Faizuddin, M., & Pradeep, A. R. (2012). Estimation of the level of tumor necrosis
factor-a in gingival crevicular fluid and serum in periodontal health & disease:
A biochemical study. Indian Journal of Dental Research, 23(3), 348–352.
Griffen, A. L., Beall, C. J., Firestone, N. D., Gross, E. L., Difranco, J. M., Hardman, J. H.,
et al. (2011). CORE: A phylogenetically-curated 16S rDNA database of the core oral
microbiome. PLoS One, 6(4), e19051.
Guzman, Y. A., Sakellari, D., Arsenakis, M., & Floudas, C. A. (2014). Proteomics for the
discovery of biomarkers and diagnosis of periodontitis: A critical review. Expert Review
of Proteomics, 11(1), 31–41.
Proteomics and Proteogenomics Approaches for Oral Diseases 155
Haririan, H., Bertl, K., Laky, M., Rausch, W. D., B€ ottcher, M., Matejka, M., et al. (2012).
Salivary and serum chromogranin A and a-amylase in periodontal health and disease.
Journal of Periodontology, 83(10), 1314–1321.
Hart, T. C., Corby, P. M., Hauskrecht, M., Hee Ryu, O., Pelikan, R., Valko, M., et al.
(2011). Identification of microbial and proteomic biomarkers in early childhood caries.
International Journal of Dentistry, 2011, 196721.
Hegde, M. N., Hegde, N. D., Ashok, A., & Shetty, S. (2014). Biochemical indicators of den-
tal caries in saliva: An in vivo study. Caries Research, 48(2), 170–173.
Heider, A., & Alt, R. (2013). VirtualArray: A R/bioconductor package to merge raw data
from different microarray platforms. BMC Bioinformatics, 14, 75.
Horst, J. A., Pieper, U., Sali, A., Zhan, L., Chopra, G., Samudrala, R., et al. (2012). Strategic
protein target analysis for developing drugs to stop dental caries. Advances in Dental
Research, 24(2), 86–93.
Houlihan, G., Gatti-Lafranconi, P., Kaltenbach, M., Lowe, D., & Hollfelder, F. (2014). An
experimental framework for improved selection of binding proteins using SNAP display.
Journal of Immunological Methods, 405, 47–56. http://dx.doi.org/10.1016/
j.jim.2014.01.006, pii: S0022-1759(14)00016-7.
Hsiao, W. W., Li, K. L., Liu, Z., Jones, C., Fraser-Liggett, C. M., & Fouad, A. F. (2012).
Microbial transformation from normal oral microbiota to acute endodontic infections.
BMC Genomics, 13, 345.
Hu, S., Arellano, M., Boontheung, P., Wang, J., Zhou, H., Jiang, J., et al. (2008). Salivary
proteomics for oral cancer biomarker discovery. Clinical Cancer Research, 14(19),
6246–6252.
Hu, S., & Wong, D. T. (2007). Oral cancer proteomics. Current Opinion in Molecular Ther-
apeutics, 9(5), 467–476.
Huang, C. M. (2004). Comparative proteomic analysis of human whole saliva. Archives of
Oral Biology, 49(12), 951–962.
Huttenhower, C., & Hofmann, O. (2010). A quick guide to large-scale genomic data min-
ing. PLoS Computational Biology, 6(5), e1000779.
Jágr, M., Eckhardt, A., Pataridis, S., & Mikšı́k, I. (2012). Comprehensive proteomic analysis
of human dentin. European Journal of Oral Sciences, 120(4), 259–268.
Jagtap, P., McGowan, T., Bandhakavi, S., Tu, Z. J., Seymour, S., Griffin, T. J., et al. (2012).
Deep metaproteomic analysis of human salivary supernatant. Proteomics, 12(7), 992–1001.
Jaiswal, G., Deo, V., Bhongade, M., & Jaiswal, S. (2011). Serum alkaline phosphatase:
A potential marker in the progression of periodontal disease in cirrhosis patients. Quin-
tessence International, 42(4), 345–348.
Jessri, M., & Farah, C. S. (2014). Next generation sequencing and its application in deci-
phering head and neck cancer. Oral Oncology, 50, 247–253. http://dx.doi.org/
10.1016/j.oraloncology.2013.12.017, pii: S1368-8375(13)00805-1.
Jiang, W., Ling, Z., Lin, X., Chen, Y., Zhang, J., Yu, J., et al. (2014). Pyrosequencing anal-
ysis of oral microbiota shifting in various caries states in childhood. Microbial Ecology,
67(4), 962–969.
Joseph, R., Rajappan, S., Nath, S. G., & Paul, B. J. (2013). Association between chronic
periodontitis and rheumatoid arthritis: A hospital-based case-control study. Rheumatology
International, 33(1), 103–109.
Jovanovic, V., Giacomelli, L., Sivozhelezov, V., Degauque, N., Lair, D., Soulillou, J. P.,
et al. (2010). AKT1 leader gene and downstream targets are involved in a rat model
of kidney allograft tolerance. Journal of Cellular Biochemistry, 111(3), 709–719.
Kalema, N., Boon, S. D., Cattamanchi, A., Davis, J. L., Andama, A., Katagira, W., et al.
(2012). Oral antimicrobial rinse to reduce mycobacterial culture contamination among
tuberculosis suspects in Uganda: A prospective study. PLoS One, 7(7), e38888.
156 Nicola Luigi Bragazzi et al.
Kanaparthy, R., & Kanaparthy, A. (2011). The changing face of dentistry: nanotechnology.
International Journal of Nanomedicine, 6, 2799–2804. http://dx.doi.org/10.2147/IJN.
S24353. Epub 2011 Nov 9.
Karlsson, M., Pålsgård, E., Wilshaw, P. R., & Di Silvio, L. (2003). Initial in vitro interaction
of osteoblasts with nano-porous alumina. Biomaterials, 24(18), 3039–3046.
Kellam, P., & Weiss, R. A. (2006). Infectogenomics: Insights from the host genome into
infectious diseases. Cell, 124(4), 695–697.
Kim, D. K., Kerman, K., Hiep, H. M., Saito, M., Yamamura, S., Takamura, Y., et al. (2008).
Label-free optical detection of aptamer-protein interactions using gold-capped oxide
nanostructures. Analytical Biochemistry, 379(1), 1–7.
Klein, M. I., Xiao, J., Lu, B., Delahunty, C. M., Yates, J. R., 3rd., & Koo, H. (2012). Strep-
tococcus mutans protein synthesis during mixed-species biofilm development by high-
throughput quantitative proteomics. PLoS One, 7(9), e45795.
Kobayashi, T., Okada, M., Ito, S., Kobayashi, D., Ishida, K., Kojima, A., et al. (2014). Assess-
ment of interleukin-6 receptor inhibition therapy on periodontal condition in patients
with rheumatoid arthritis and chronic periodontitis. Journal of Periodontology, 85(1),
57–67.
Kobeissy, F. H., Gulbakan, B., Alawieh, A., Karam, P., Zhang, Z., Guingab-Cagmat, J. D.,
et al. (2014). Post-genomics nanotechnology is gaining momentum: Nanoproteomics
and applications in life sciences. Omics: A Journal of Integrative Biology, 18(2), 111–131.
Kodama, Y., Kaminuma, E., Saruhashi, S., Ikeo, K., Sugawara, H., Tateno, Y., et al. (2010).
Biological databases at DNA Data Bank of Japan in the era of next-generation sequencing
technologies. Advances in Experimental Medicine and Biology, 680, 125–135.
Kornman, K. S., & Duff, G. W. (2012). Personalized medicine: Will dentistry ride the wave
or watch from the beach? Journal of Dental Research, 91(7 Suppl), 8S–11S.
Krishna Prasad, R. B., Sharma, A., & Babu, H. M. (2013). An insight into salivary markers in
oral cancer. Dental Research Journal, 10(3), 287–295.
Kuboniwa, M., Tribble, G. D., Hendrickson, E. L., Amano, A., Lamont, R. J., &
Hackett, M. (2012). Insights into the virulence of oral biofilms: Discoveries from pro-
teomics. Expert Review of Proteomics, 9(3), 311–323.
Laine, M. L., Moustakis, V., Koumakis, L., Potamias, G., & Loos, B. G. (2013). Modeling
susceptibility to periodontitis. Journal of Dental Research, 92(1), 45–50.
Lamster, I. B., & Ahlo, J. K. (2007). Analysis of gingival crevicular fluid as applied to the
diagnosis of oral and systemic diseases. Annals of the New York Academy of Sciences,
1098, 216–229.
Le, H. S., Oltvai, Z. N., & Bar-Joseph, Z. (2010). Cross-species queries of large gene expres-
sion databases. Bioinformatics, 26(19), 2416–2423.
Lee, S. Y., Park, H. R., Cho, N. H., Choi, Y. P., Rha, S. Y., Park, S. W., et al. (2013).
Identifying genes related to radiation resistance in oral squamous cell carcinoma cell lines.
International Journal of Oral and Maxillofacial Surgery, 42(2), 169–176.
Lemos, J. A., Abranches, J., & Burne, R. A. (2005). Responses of cariogenic streptococci to
environmental stresses. Current Issues in Molecular Biology, 7(1), 95–107.
Lemos, J. A., & Burne, R. A. (2008). A model of efficiency: Stress tolerance by Streptococcus
mutans. Microbiology, 154(Pt 11), 3247–3255.
Levine, A. E., & Steffen, D. L. (2001). OrCGDB: A database of genes involved in oral cancer.
Nucleic Acids Research, 29(1), 300–302.
Li, Y., St John, M. A., Zhou, X., Kim, Y., Sinha, U., Jordan, R. C., et al. (2004). Salivary
transcriptome diagnostics for oral cancer detection. Clinical Cancer Research, 10(24),
8442–8450.
Li, J., Wang, W., Wang, Y., & Zeng, A. P. (2013). Two-dimensional gel-based proteomic of
the caries causative bacterium Streptococcus mutans UA159 and insight into the inhib-
itory effect of carolacton. Proteomics, 13(23–24), 3470–3477.
Proteomics and Proteogenomics Approaches for Oral Diseases 157
Liu, B., Faller, L. L., Klitgord, N., Mazumdar, V., Ghodsi, M., Sommer, D. D., et al. (2012).
Deep sequencing of the oral microbiome reveals signatures of periodontal disease. PLoS
One, 7(6), e37919.
Mallineni, S. K., Nuvvula, S., Matinlinna, J. P., Yiu, C. K., & King, N. M. (2013). Biocom-
patibility of various dental materials in contemporary dentistry: A narrative insight. Jour-
nal of Investigative and Clinical Dentistry, 4(1), 9–19.
Mantri, S. S., & Mantri, S. P. (2013). The nano era in dentistry. Journal of Natural Science,
Biology, and Medicine, 4(1), 39–44.
Manzano-Román, R., Dı́az-Martı́n, V., González-González, M., Matarraz, S., Álvarez-
Prado, A. F., LaBaer, J., et al. (2012). Self-assembled protein arrays from an Ornithodoros
moubata salivary gland expression library. Journal of Proteome Research, 11(12), 5972–5982.
Marconcini, S., Covani, U., Barone, A., Vittorio, O., Curcio, M., Barbuti, S., et al. (2011).
Real-time quantitative polymerase chain reaction analysis of patients with refractory
chronic periodontitis. Journal of Periodontology, 82(7), 1018–1024.
Marconcini, S., Genovesi, A. M., Marchisio, O., Gelpi, F., Barone, A., Corega, C., et al.
(2014). In vivo study of titanium healing screws surface modifications after different
debridment procedure. Minerva Stomatologica, (Epub ahead of print).
Marimuthu, A., Chavan, S., Sathe, G., Sahasrabuddhe, N. A., Srikanth, S. M., Renuse, S.,
et al. (2013). Identification of head and neck squamous cell carcinoma biomarker can-
didates through proteomic analysis of cancer cell secretome. Biochimica et Biophysica Acta,
1834(11), 2308–2316.
Masuda, H., & Fukuda, K. (1995). Ordered metal nanohole arrays made by a two-step rep-
lication of honeycomb structures of anodic alumina. Science, 268, 1466–1468.
Matse, J. H., Yoshizawa, J., Wang, X., Elashoff, D., Bolscher, J. G., Veerman, E. C., et al.
(2013). Discovery and prevalidation of salivary extracellular microRNA biomarkers
panel for the noninvasive detection of benign and malignant parotid gland tumors. Clin-
ical Cancer Research, 19(11), 3032–3038.
Mazumdar, V., Snitkin, E. S., Amar, S., & Segrè, D. (2009). Metabolic network model of a
human oral pathogen. Journal of Bacteriology, 191(1), 74–90.
McCartan, B. E., & Healy, C. M. (2008). The reported prevalence of oral lichen planus:
A review and critique. Journal of Oral Pathology & Medicine, 37(8), 447–453.
Meurman, J. H., Janket, S. J., Qvarnstr€ om, M., & Nuutinen, P. (2003). Dental infections and
serum inflammatory markers in patients with and without severe heart disease. Oral
Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics, 96(6), 695–700.
Miersch, S., Bian, X., Wallstrom, G., Sibani, S., Logvinenko, T., Wasserfall, C. H., et al.
(2013). Serological autoantibody profiling of type 1 diabetes by protein arrays. Journal
of Proteomics, 94, 486–496.
Mitra, S., Das, S., Ghosal, S., & Chakrabarti, J. (2012). HNOCDB: A comprehensive data-
base of genes and miRNAs relevant to head and neck and oral cancer. Oral Oncology,
48(2), 117–119.
Montor, W. R., Huang, J., Hu, Y., Hainsworth, E., Lynch, S., Kronish, J. W., et al. (2009).
Genome-wide study of Pseudomonas aeruginosa outer membrane protein immunoge-
nicity using self-assembling protein microarrays. Infection and Immunity, 77(11),
4877–4886.
Moreau, Y., & Tranchevent, L. C. (2012). Computational tools for prioritizing candidate
genes: Boosting disease gene discovery. Nature Reviews. Genetics, 13(8), 523–536.
Nakano, Y., Shibata, Y., Kawada, M., Kojima, M., Fukamachi, H., Shibata, Y., et al. (2005).
A searchable database for proteomes of oral microorganisms. Oral Microbiology and Immu-
nology, 20(6), 344–348.
Nan, J., Brostromer, E., Liu, X. Y., Kristensen, O., & Su, X. D. (2009). Bioinformatics and
structural characterization of a hypothetical protein from Streptococcus mutans: Impli-
cation of antibiotic resistance. PLoS One, 4(10), e7245.
158 Nicola Luigi Bragazzi et al.
Nand, A., Gautam, A., Pérez, J. B., Merino, A., & Zhu, J. (2012). Emerging technology of
in situ cell free expression protein microarrays. Protein & Cell, 3(2), 84–88.
NCBI Resource Coordinators (2014). Database resources of the National Center for Bio-
technology Information. Nucleic Acids Research, 42(1), D7–D17.
Nibali, L., Donos, N., & Henderson, B. (2009). Periodontal infectogenomics. Journal of Med-
ical Microbiology, 58(Pt 10), 1269–1274.
Nicolini, C. (2006). Nanogenomics for medicine. Nanomedicine (London, England), 1(2),
147–152.
Nicolini, C., Spera, R., Stura, E., Fiordoro, S., & Giacomelli, L. (2006). Gene expression in
the cell cycle of human T-lymphocytes: II Experimental determination by DNASER
technology. Journal of Cellular Biochemistry, 97(5), 1151–1159.
Nicolini, C. (2010). Nanogenomics in medicine. Wiley Interdisciplinary Reviews. Nanomedicine
and Nanobiotechnology, 2(1), 59–76.
Nicolini, C., Adami, M., Sartore, M., Bragazzi, N. L., Bavastrello, V., Spera, R., et al. (2012).
Prototypes of newly conceived inorganic and biological sensors for health and environ-
mental applications. Sensors (Basel, Switzerland), 12(12), 17112–17127.
Nicolini, C., Bragazzi, N., & Pechkova, E. (2012). Nanoproteomics enabling personalized
nanomedicine. Advanced Drug Delivery Reviews, 64(13), 1522–1531.
Nicolini, C., Bragazzi, N., & Pechkova, E. (2013). From nanobiotechnology to organic and
biological monitoring of health and environment for biosafety. Journal of Bioanalysis &
Biomedicine, 5, 108–117.
Nicolini, C., Correia, T. B., Stura, E., Larosa, C., Spera, R., & Pechkova, E. (2013). Atomic
force microscopy and anodic porous allumina of nucleic acid programmable protein
arrays. Recent Patents on Biotechnology, 7(2), 112–121.
Nicolini, C., & Pechkova, E. (2010a). An overview of nanotechnology-based functional pro-
teomics for cancer and cell cycle progression. Anticancer Research, 30(6), 2073–2080.
Nicolini, C., & Pechkova, E. (2010b). Nanoproteomics for nanomedicine. Nanomedicine
(London, England), 5(5), 677–682.
Nicolini, C., Singh, M., Spera, R., & Felli, L. (2013). Analysis of gene expression on anodic
porous alumina microarrays. Bioengineered, 4(5), 332–337.
Nicolini, C., Spera, R., Festa, F., Belmonte, L., Chong, S., Pechkova, E., et al. (2013). Mass
spectrometry and florescence analysis of SNAP-NAPPA arrays expressed using E. coli
cell_free expression system. Journal of Nanomedicine & Nanotechnology, 4(5), 181–195.
Ogawa, Y., Miura, Y., Harazono, A., Kanai-Azuma, M., Akimoto, Y., Kawakami, H., et al.
(2011). Proteomic analysis of two types of exosomes in human whole saliva. Biological &
Pharmaceutical Bulletin, 34(1), 13–23.
Okada, M., Kobayashi, T., Ito, S., Yokoyama, T., Abe, A., Murasawa, A., et al. (2013). Peri-
odontal treatment decreases levels of antibodies to Porphyromonas gingivalis and citrul-
line in patients with rheumatoid arthritis and periodontitis. Journal of Periodontology,
84(12), e74–e84.
Orlando, B., Bragazzi, N., & Nicolini, C. (2013). Bioinformatics and systems biology analysis
of genes network involved in OLP (Oral Lichen Planus) pathogenesis. Archives of Oral
Biology, 58(6), 664–673.
Ozak, S. T., & Ozkan, P. (2013). Nanotechnology and dentistry. European Journal of Dentistry,
7(1), 145–151.
Patel, S. P., & Raju, P. A. (2013). Resistin in serum and gingival crevicular fluid as a marker of
periodontal inflammation and its correlation with single-nucleotide polymorphism in
human resistin gene at -420. Contemporary Clinical Dentistry, 4(2), 192–197.
Pechkova, E., Chong, S., Tripathi, S., & Nicolini, C. (2010). Cell free expression and APA
for NAPPA and protein crystallography: Functional proteomics and nanotechnology-
based microarrays. In C. Nicolini & J. LaBaer (Eds.), Pan stanford series on nanobio-
technology 2 (pp. 121–147). London - New York - Singapore: Thomson ISI Web
of Science.
Proteomics and Proteogenomics Approaches for Oral Diseases 159
Petersen, P. E. (2009). Oral cancer prevention and control—The approach of the World
Health Organization. Oral Oncology, 45(4–5), 454–460.
Peterson, S. N., Snesrud, E., Schork, N. J., & Bretz, W. A. (2011). Dental caries pathoge-
nicity: A genomic and metagenomic perspective. International Dental Journal, 61(Suppl 1),
11–22.
Pham, T. K., Roy, S., Noirel, J., Douglas, I., Wright, P. C., & Stafford, G. P. (2010).
A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen
Tannerella forsythia. Proteomics, 10(17), 3130–3141.
Pradeep, A. R., Kumari, M., Kalra, N., & Priyanka, N. (2013). Correlation of MCP-4 and
high-sensitivity C-reactive protein as a marker of inflammation in obesity and chronic
periodontitis. Cytokine, 61(3), 772–777.
Pradeep, A. R., Raghavendra, N. M., Prasad, M. V., Kathariya, R., Patel, S. P., & Sharma, A.
(2011). Gingival crevicular fluid and serum visfatin concentration: Their relationship in
periodontal health and disease. Journal of Periodontology, 82(9), 1314–1319.
Preza, D., Thiede, B., Olsen, I., & Grinde, B. (2009). The proteome of the human parotid
gland secretion in elderly with and without root caries. Acta Odontologica Scandinavica,
67(3), 161–169.
Racapé, M., Bragazzi, N., Sivozhelezov, V., Danger, R., Pechkova, E., Duong Van
Huyen, J. P., et al. (2012). SMILE silencing and PMA activation gene networks in HeLa
cells: Comparison with kidney transplantation gene networks. Journal of Cellular Biochem-
istry, 113(6), 1820–1832.
Raghavendra, N. M., Pradeep, A. R., Kathariya, R., Sharma, A., Rao, N. S., & Naik, S. B.
(2012). Effect of non surgical periodontal therapy on gingival crevicular fluid and
serum visfatin concentration in periodontal health and disease. Disease Markers, 32(6),
383–388.
Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, S., Rosen, B., Lau, A. Y., et al.
(2004). Self-assembling protein microarrays. Science, 305(5680), 86–90.
Rappaport, N., Nativ, N., Stelzer, G., Twik, M., Guan-Golan, Y., Stein, T. I., et al. (2013).
MalaCards: An integrated compendium for diseases and their annotation. Database,
2013, bat018.
Rappuoli, R. (2000). Reverse vaccinology. Current Opinion in Microbiology, 3(5), 445–450.
Razzouk, S., & Termechi, O. (2013). Host genome, epigenome, and oral microbiome inter-
actions: Toward personalized periodontal therapy. Journal of Periodontology, 84(9),
1266–1271.
Reddy, P. J., Jain, R., Paik, Y. K., Downey, R., Ptolemy, A. S., Ozdemir, V., et al. (2011).
Personalized medicine in the age of pharmacoproteomics: A close up on India and need
for social science engagement for responsible innovation in post-proteomic biology.
Current Pharmacogenomics and Personalized Medicine, 9(1), 67–75.
Reshmi, G., Charles, S., James, P., Jijith, V. S., Prathibha, R., Ramachandran, S., et al.
(2012). OrCa-dB: A complete catalogue of molecular and clinical information in oral
carcinogenesis. Oral Oncology, 48(6), e19.
Richards, D. (2013). Oral diseases affect some 3.9 billion people. Evidence-Based Dentistry,
14(2), 35.
Riley, D. J., Bavastrello, V., Covani, U., Barone, A., & Nicolini, C. (2005). An in-vitro study
of the sterilization of titanium dental implants using low intensity UV-radiation. Dental
Materials, 21(8), 756–760.
Rolfs, A., Montor, W. R., Yoon, S. S., Hu, Y., Bhullar, B., Kelley, F., et al. (2008). Pro-
duction and sequence validation of a complete full length ORF collection for the path-
ogenic bacterium Vibrio cholerae. Proceedings of the National Academy of Sciences of the
United States of America, 105(11), 4364–4369.
Rosa, N., Correia, M. J., Arrais, J. P., Lopes, P., Melo, J., Oliveira, J. L., et al. (2012). From
the salivary proteome to the OralOme: Comprehensive molecular oral biology. Archives
of Oral Biology, 57(7), 853–864.
160 Nicola Luigi Bragazzi et al.
Ross, B. C., Czajkowski, L., Hocking, D., Margetts, M., Webb, E., Rothel, L., et al. (2001).
Identification of vaccine candidate antigens from a genomic analysis of Porphyromonas
gingivalis. Vaccine, 19(30), 4135–4142.
Ruhl, S. (2012). The scientific exploration of saliva in the post-proteomic era: From database
back to basic function. Expert Review of Proteomics, 9(1), 85–96.
Rustici, G., Kolesnikov, N., Brandizi, M., Burdett, T., Dylag, M., Emam, I., et al. (2013).
ArrayExpress update—Trends in database growth and links to data analysis tools. Nucleic
Acids Research, 41(Database issue), D987–D990.
Salerno, M., Caneva-Soumetz, F., Pastorino, L., Patra, N., Diaspro, A., & Ruggiero, C.
(2013). Adhesion and proliferation of osteoblast-like cells on anodic porous alumina
substrates with different morphology. IEEE Transactions on Nanobioscience, 12(2), 106–111.
Salerno, M., Giacomelli, L., & Larosa, C. (2011). Biomaterials for the programming of cell
growth in oral tissues: The possible role of APA. Bioinformation, 5(7), 291–293.
Sbordone, L., Sbordone, C., Filice, N., Menchini-Fabris, G., Baldoni, M., & Toti, P. (2009).
Gene clustering analysis in human osseous remodeling. Journal of Periodontology, 80(12),
1998–2009.
Schleyer, T. K. (2003). Dental informatics: An emerging biomedical informatics discipline.
Journal of Dental Education, 67(11), 1193–1200.
Schleyer, T., Mattsson, U., Nı́ Rı́ordáin, R., Brailo, V., Glick, M., Zain, R. B., et al. (2011).
Advancing oral medicine through informatics and information technology: A proposed
framework and strategy. Oral Diseases, 17(Suppl 1), 85–94.
Schwarzberg, K., Le, R., Bharti, B., Lindsay, S., Casaburi, G., Salvatore, F., et al. (2014). The
personal human oral microbiome obscures the effects of treatment on periodontal dis-
ease. PLoS One, 9(1), e86708.
Sharma, A., Pradeep, A. R., Raghavendra, N. M., Arjun, P., & Kathariya, R. (2012). Gin-
gival crevicular fluid and serum cystatin c levels in periodontal health and disease. Disease
Markers, 32(2), 101–107.
Shenar, N., Martinez, J., & Enjalbal, C. (2008). Laser desorption/ionization mass spectrom-
etry on porous silica and alumina for peptide mass fingerprinting. Journal of the American
Society for Mass Spectrometry, 19(5), 632–644.
Sibani, S., & LaBaer, J. (2011). Immunoprofiling using NAPPA protein microarrays. Methods
in Molecular Biology, 723, 149–161.
Silverman, S., Jr., Gorsky, M., & Lozada-Nur, F. (1985). A prospective follow-up study of
570 patients with oral lichen planus: Persistence, remission, and malignant association.
Oral Surgery, Oral Medicine, and Oral Pathology, 60(1), 30–34.
Sivozhelezov, V., Braud, C., Giacomelli, L., Pechkova, E., Giral, M., Soulillou, J. P., et al.
(2008). Immunosuppressive drug-free operational immune tolerance in human kidney
transplants recipients. Part II. Non-statistical gene microarray analysis. Journal of Cellular
Biochemistry, 103(6), 1693–1706.
Sivozhelezov, V., Giacomelli, L., Tripathi, S., & Nicolini, C. (2006). Gene expression in the
cell cycle of human T lymphocytes: I. Predicted gene and protein networks. Journal of
Cellular Biochemistry, 97(5), 1137–1150.
Sivozhelezov, V., Spera, R., Giacomelli, L., Hainsworth, E., Labaer, J., Bragazzi, N. L., et al.
(2009). Bioinformatics and fluorescence DNASER for NAPPA studies on cell transfor-
mation and cell cycle. In Functional Proteomics and Nanotechnology-Based Microarrays: Vol. 2
(pp. 31–59).
Skates, S. J., Gillette, M. A., LaBaer, J., Carr, S. A., Anderson, L., Liebler, D. C., et al. (2013).
Statistical design for biospecimen cohort size in proteomics-based biomarker discovery
and verification studies. Journal of Proteome Research, 12(12), 5383–5394.
Song, Y., Ju, Y., Morita, Y., & Song, G. (2013). Effect of the nanostructure of porous alu-
mina on growth behavior of MG63 osteoblast-like cells. Journal of Bioscience and Bioengi-
neering, 116(4), 509–515.
Proteomics and Proteogenomics Approaches for Oral Diseases 161
Spera, R., Badino, F., Hainsworth, E., Fuentes, M., Srivastava, S., LaBaer, J., et al. (2010).
Label free detection of NAPPA via mass spectrometry. In J. LaBaer & C. Nicolini (Eds.),
Functional Proteomics and Nanotechnology-Based Microarrays (pp. 61–78). Pan Stanford
Publishing (Chapter).
Spera, R., Correia, T. T. B., & Nicolini, C. (2013a). NAPPA based nanogravimetric
biosensor: Preliminary characterization. Sensors and Actuators, B: Chemical, 182,
682–688.
Spera, R., Festa, F., Bragazzi, N. L., Pechkova, E., LaBaer, J., & Nicolini, C. (2013b). Con-
ductometric monitoring of protein-protein interactions. Journal of Proteome Research,
12(12), 5535–5547.
Spera, R., Labaer, J., & Nicolini, C. (2011). MALDI-TOF characterization of NAPPA-
generated proteins. Journal of Mass Spectrometry, 46(9), 960–965.
Stelzer, G., Dalah, I., Stein, T. I., Satanower, Y., Rosen, N., Nativ, N., et al. (2011). In-silico
human genomics with GeneCards. Human Genomics, 5(6), 709–717.
Strimbu, K., & Tavel, J. A. (2010). What are biomarkers? Current Opinion in HIV and AIDS,
5(6), 463–466.
Stura, E., Bruzzese, D., Valerio, F., Grasso, V., Perlo, P., & Nicolini, C. (2007). Anodic
porous alumina as mechanical stability enhancer for LDL-cholesterol sensitive electrodes.
Biosensors & Bioelectronics, 23(5), 655–660.
Sundar, N. M., Krishnan, V., Krishnaraj, S., Hemalatha, V. T., & Alam, M. N. (2013). Com-
parison of the salivary and the serum nitric oxide levels in chronic and aggressive peri-
odontitis: A biochemical study. Journal of Clinical and Diagnostic Research, 7(6),
1223–1227.
Suzuki, A., Takai-Igarashi, T., Numabe, Y., & Tanaka, H. (2009). Development of a data-
base and ontology for pathogenic pathways in periodontitis. In Silico Biology, 9(4),
233–243.
Thanawastien, A., Montor, W. R., Labaer, J., Mekalanos, J. J., & Yoon, S. S. (2009). Vibrio
cholerae proteome-wide screen for immunostimulatory proteins identifies phos-
phatidylserine decarboxylase as a novel Toll-like receptor 4 agonist. PLoS Pathogen,
5(8), e1000556.
Thorat, M., Pradeep, A. R., & Garg, G. (2010). Correlation of levels of oncostatin
M cytokine in crevicular fluid and serum in periodontal disease. International Journal of
Oral Science, 2(4), 198–207.
Tung, C. L., Lin, S. T., Chou, H. C., Chen, Y. W., Lin, H. C., Tung, C. L., et al. (2013).
Proteomics-based identification of plasma biomarkers in oral squamous cell carcinoma.
Journal of Pharmaceutical and Biomedical Analysis, 75, 7–17.
Wade, W. G. (2013). The oral microbiome in health and disease. Pharmacological Research,
69(1), 137–143.
Wang, Y., Xia, X., & Guo, Y. (2005). Porous anodic alumina membrane as a sample support
for MALDI-TOF MS analysis of salt-containing proteins. Journal of the American Society for
Mass Spectrometry, 16(9), 1488–1492.
Wang, Q., Jia, P., Cuenco, K. T., Feingold, E., Marazita, M. L., Wang, L., et al. (2013).
Multi-dimensional prioritization of dental caries candidate genes and its enriched dense
network modules. PLoS One, 8(10), e76666.
Wiebe, C. B., & Putnins, E. E. (2000). The periodontal disease classification system of the
American Academy of Periodontology—An update. Journal of the Canadian Dental Asso-
ciation, 66(11), 594–597.
Wong, D. (Ed.), (2009). Salivary Diagnostics (320 pages). Wiley-Blackwell. ISBN: 978-0-
8138-0664-8.
Wright, C., Sibani, S., Trudgian, D., Fischer, R., Kessler, B., LaBaer, J., et al. (2012). Detec-
tion of multiple autoantibodies in patients with ankylosing spondylitis using nucleic acid
programmable protein arrays. Molecular & Cellular Proteomics, 11(2), M9.00384.
162 Nicola Luigi Bragazzi et al.
Xie, G., Chain, P. S., Lo, C. C., Liu, K. L., Gans, J., Merritt, J., et al. (2010). Community and
gene composition of a human dental plaque microbiota obtained by metagenomic
sequencing. Molecular Oral Microbiology, 25(6), 391–405.
Xue, V., Burdett, T., Lukk, M., Taylor, J., Brazma, A., & Parkinson, H. (2012). Mage-
Comet—Web application for harmonizing existing large-scale experiment descriptions.
Bioinformatics, 28(10), 1402–1403.
Yang, L. L., Liu, X. Q., Liu, W., Cheng, B., & Li, M. T. (2006). Comparative analysis of
whole saliva proteomes for the screening of biomarkers for oral lichen planus. Inflamma-
tion Research, 55(10), 405–407.
Yu, W. H., Hu, H., Zhou, Q., Xia, Y., & Amar, S. (2010). Bioinformatics analysis of mac-
rophages exposed to Porphyromonas gingivalis: Implications in acute vs. chronic infec-
tions. PLoS One, 5(12), e15613.
Zainal-Abidin, Z., Veith, P. D., Dashper, S. G., Zhu, Y., Catmull, D. V., Chen, Y. Y., et al.
(2012). Differential proteomic analysis of a polymicrobial biofilm. Journal of Proteome
Research, 11(9), 4449–4464.
Zandparsa, R. (2014). Latest biomaterials and technology in dentistry. Dental Clinics of North
America, 58(1), 113–134.
Zehetbauer, S., Wojahn, T., Hiller, K. A., Schmalz, G., & Ruhl, S. (2009). Resemblance of
salivary protein profiles between children with early childhood caries and caries-free
controls. European Journal of Oral Sciences, 117(4), 369–373.
Zhan, Y., Zhang, R., Lv, H., Song, X., Xu, X., Chai, L., et al. (2014). Prioritization of can-
didate genes for periodontitis using multiple computational tools. Journal of Periodontology,
(Epub ahead of print).
Zhang, A., Sun, H., Wang, P., & Wang, X. (2013). Salivary proteomics in biomedical
research. Clinica Chimica Acta, 415, 261–265.
Zheng, J., Stoyanovich, J., Manduchi, E., Liu, J., & Stoeckert, C. J., Jr. (2011).
AnnotCompute: Annotation-based exploration and meta-analysis of genomics experi-
ments. Database, 2011, bar045.
Zijnge, V., Kieselbach, T., & Oscarsson, J. (2012). Proteomics of protein secretion by
Aggregatibacter actinomycetemcomitans. PLoS One, 7(7), e41662.
CHAPTER FIVE
Advances in Nanocrystallography
as a Proteomic Tool
Eugenia Pechkova*,†, Nicola Luigi Bragazzi*,†,{, Claudio Nicolini*,†,},1
*Nanobiotechnology and Biophysics Laboratories (NBL), Department of Experimental Medicine (DIMES),
University of Genoa, Genoa, Italy
†
Nanoworld Institute Fondazione ELBA Nicolini (FEN), Pradalunga, Bergamo, Italy
{
School of Public Health, Department of Health Sciences (DISSAL), University of Genoa, Genoa, Italy
}
Biodesign Institute, Arizona State University, Tempe, Arizona, USA
1
Corresponding author: e-mail address: [email protected]
Contents
1. Introduction 164
2. Langmuir–Blodgett (LB)-Based Crystallization 166
3. Comparison of LB-Based Crystallization with Other Techniques 168
4. Fourier Transform Infrared (FTIR) Spectroscopy for Investigating LB-Films 170
5. Raman Spectroscopy 171
6. Laser-Induced Microdissection and Microfragmentation 172
7. Micrograzing-Incidence X-Ray Scattering Angle (m-GISAXS) 172
8. In Silico Simulations 176
9. Bioinformatics 177
10. Molecular Dynamics 178
11. Clinically Relevant Proteins 180
11.1 GroEL 180
11.2 Casein kinase 2 181
11.3 Cytochrome P-450 side-chain cleavage 181
11.4 Rhodopsin 182
11.5 Globins 183
11.6 Insulin 183
12. Conclusions 184
References 185
Abstract
In order to overcome the difficulties and hurdles too much often encountered in crys-
tallizing a protein with the conventional techniques, our group has introduced the inno-
vative Langmuir–Blodgett (LB)-based crystallization, as a major advance in the field of
both structural and functional proteomics, thus pioneering the emerging field of the
so-called nanocrystallography or nanobiocrystallography. This approach uniquely com-
bines protein crystallography and nanotechnologies within an integrated, coherent
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 163
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00005-1
164 Eugenia Pechkova et al.
framework that allows one to obtain highly stable protein crystals and to fully charac-
terize them at a nano- and subnanoscale.
A variety of experimental techniques and theoretical/semi-theoretical approaches,
ranging from atomic force microscopy, circular dichroism, Raman spectroscopy and
other spectroscopic methods, microbeam grazing-incidence small-angle X-ray scatter-
ing to in silico simulations, bioinformatics, and molecular dynamics, has been exploited
in order to study the LB-films and to investigate the kinetics and the main features of
LB-grown crystals.
When compared to classical hanging-drop crystallization, LB technique appears
strikingly superior and yields results comparable with crystallization in microgravity
environments.
Therefore, the achievement of LB-based crystallography can have a tremendous
impact in the field of industrial and clinical/therapeutic applications, opening new per-
spectives for personalized medicine. These implications are envisaged and discussed in
the present contribution.
1. INTRODUCTION
Since the birth of crystallography in 1840 (Giegé, 2013), over the past
decades, a remarked progress in the field of protein structure determination has
been achieved thanks to advancements in X-ray crystallography combined
with the brighter and highly focused, third-generation synchrotron
(Belmonte, Pechkova, Tripathi, Scudieri, & Nicolini, 2012; Riekel, 2004;
Riekel, Burghammer, & Schertler, 2005). This unique method based on a
combined and integrated approach is likely to remain the most important
method for the determination of protein structure in the foreseeable future
(Belmonte et al., 2012). In comparison with other methods, such as (cryo)elec-
tron microscopy, nuclear magnetic resonance, solution scattering, and neu-
tron diffraction, X-ray crystallography is indeed the most utilized approach
for obtaining a full atom detailed structure of the investigated protein.
However, in the literature, there is a remarked lack of both structural and
functional studies devoted to the investigation and characterization of ther-
apeutically and clinically relevant proteins, considering that at least 60% of
the commercially available drugs target membrane proteins (Drews, 2000),
which are instead scarcely represented on the Protein Data Bank (PDB)
(Kang, Lee, & Drew, 2013; Ubarretxena-Belandia & Stokes, 2010). This
is because of the initial reluctance of the protein to being crystallized and
the difficulties often encountered while trying to develop standardized pro-
tocols, which would shift crystallography from an art to a science (Chayen,
2004; Chayen & Saridakis, 2008; Helliwell & Chayen, 2007). These hurdles
Advances in Nanocrystallography as a Proteomic Tool 165
2. LANGMUIR–BLODGETT (LB)-BASED
CRYSTALLIZATION
LB nanotemplate crystallization method has proved to give prominent
results in target proteins crystallization, such as thaumatin, a 207-amino acid,
22.2-kDa sweet-tasting protein, obtained from the African plant
Thaumatococcus daniellii Bennett (Gebhardt, Pechkova, Riekel, & Nicolini,
2010; Pechkova, Gebhardt, Riekel, & Nicolini, 2010; Pechkova,
Scudieri, Belmonte, & Nicolini, 2012; Pechkova, Sivozhelezov,
Belmonte, & Nicolini, 2012), which can be used as a low-calorie sweetener
and flavor modifier. Other proteins that have been tested for crystallization
are lysozyme, EC 3.2.1.17, a 14.7-kDa protein, of Gallus gallus (Hen egg
white lysozyme) (Pechkova & Nicolini, 2002; Pechkova, Roth, et al.,
2005; Pechkova, Vasile, et al., 2005; Pechkova, Sartore, Giacomelli, &
Nicolini, 2007; Pechkova, Sivozhelezov, & Nicolini, 2007), ribonuclease
A, EC 3.1.27.5, a small 124-amino acid, 13.7-kDa RNAse from Bos taurus
(Pechkova, Scudieri, et al., 2012; Pechkova, Sivozhelezov, et al., 2012),
thermolysin, EC 3.4.24.27, a 34.6-kDa enzyme of Bacillus thermoproteolyticus
(Pechkova, Scudieri, et al., 2012; Pechkova, Sivozhelezov, et al., 2012), and
proteinase K, EC 3.4.21.64, of Engyodontium album, formerly known as
Tritirachium album (Pechkova, Tripathi, & Nicolini, 2009; Pechkova,
Advances in Nanocrystallography as a Proteomic Tool 167
water, purified and filtered with Milli-Q system, 18.2 MO cm). Later, uti-
lizing the Langmuir–Schaefer (LS) method (horizontal lift), the floating pro-
tein thin film is transferred onto the surface of a solid substrate (such as
siliconized circular glass or glass wafers, washed in water, and dried in a gas-
eous nitrogen flux). LS technique consists of making the prepared substrate
horizontally touching the monolayer or multilayer, in such a way that the
layer transfers itself onto the substrate surface.
In case of multilayer deposition, the regularity and uniformity can be
controlled using nanogravimetric measurements and verifying by comput-
ing the area per molecule of the proper packing density.
The trough is connected to a personal computer that, being equipped
with dedicated software, enables the measuring and recording of surface
phenomena due to Teflon barriers movement and compression, after prop-
erly stabilizing the paper Wilhelmy plate.
The advantages of using this nanobiotechnology are different: they
include the accelerated nucleation and growth of protein crystals
(Pechkova & Nicolini, 2002), their higher quality both in terms of X-ray
diffraction and radiation stability when employing the high energy X-ray
source and focused beans, such as the third-generation synchrotrons and
the microdiffraction beamlines (Belmonte et al., 2012; Pechkova et al.,
2004). This could be due to enhanced mechanical properties of the protein
crystals, as we review and describe in detail in the following sections. More-
over, LB-grown crystals are larger, have a more regular and clearly defined
shape, and more perfect domains (Pechkova & Nicolini, 2010; Riekel et al.,
2011) (Fig. 5.2).
and CID, and microbatch) ( Judge, Snell, & van der Woerd, 2005;
Khurshid & Chayen, 2006; Otálora, Gavira, Ng, & Garcı́a-Ruiz, 2009;
Vergara, Lorber, Sauter, Giegé, & Zagari, 2005). Very recently, also seeding
and heterogenous nucleations have been applied in space (Sch€ ope &
Wette, 2011).
Differences in protein crystal formation between classical hanging-drop
and LB have been established by a variety of methods including both ex situ
and in situ GISAXS (Gebhardt et al., 2010; Nicolini & Pechkova, 2006;
Nicolini, Belmonte, et al., 2013; Nicolini, Bragazzi, et al., 2013;
Nicolini, Bruzzese, et al., 2013; Nicolini, Correia, et al., 2013; Nicolini,
Belmonte, Riekel, Koenig, & Pechkova, 2014; Nicolini, Bragazzi,
Pechkova, & Lazzari, 2014; Pechkova & Nicolini, 2006, 2011; Pechkova,
Roth, et al., 2005; Pechkova, Vasile, et al., 2005; Pechkova, Tripathi, &
Nicolini, 2009; Pechkova, Tripathi, Ravelli, et al., 2009; Pechkova et al.,
2010), laser microdissection combined with nano- and microfocus
beamlines (Nicolini, Belmonte, et al., 2014; Nicolini, Bragazzi, et al.,
2014; Riekel et al., 2011), Raman spectroscopy (Nicolini, Belmonte,
et al., 2013; Nicolini, Bragazzi, et al., 2013; Nicolini, Bruzzese, et al.,
2013; Nicolini, Correia, et al., 2013), and atomic force microscopy
(Pechkova, Sartore, et al., 2007; Pechkova, Sivozhelezov, et al., 2007). In
the following paragraphs and sections, we overview the main conclusions
originated from these experiments.
5. RAMAN SPECTROSCOPY
Raman spectroscopy (named after the Indian physicist Sir
Chandrasekhara Venkata Raman) is an advanced spectroscopic technique
that enables to observe and measure vibrational, rotational, and other
low-frequency modes of a given system, relying on inelastic scattering or
Raman scattering.
Raman spectroscopy is attractive as a potential diagnostic technology
because it requires no extrinsic labeling, is not limited by masking water con-
tributions, and is inherently a multiplexing technique. Raman-based mea-
surements of biological samples have already been exploited for the
identification of molecular-specific markers for disease detection and moni-
toring (Turzhitsky et al., 2014). The use of fiber optic technology coupled
with Raman spectroscopy is ideal for application to aqueous solutions, either
with or without LB nanotemplate. Raman is expected to evaluate the protein
concentration changes in vapor diffusion hanging-drop method with and
without LB nanotemplate. Acquired Raman spectra have previously been
subjected to quantitative infrared partial least squares (PLS) models with
remarkable success. The PLS model generated correlates the spectral region
from 2700 to 3600 cm1 with the concentration (g/ml) of lysozyme. This
spectral region encompasses vibrations due to the protein C–H stretches cen-
tered at 2950 cm1 and the water O–H stretches centered at 3230 cm1.
On the basis of our Raman spectroscopic analysis (Nicolini, Belmonte,
et al., 2013; Nicolini, Bragazzi, et al., 2013; Nicolini, Bruzzese, et al., 2013;
Nicolini, Correia, et al., 2013), we suggest that LB-assisted crystal growth
with time is accompanied by:
1. Formation of disulfide bonds S6–S127/S30–S115 that brings Trp123
into new position and facilitates vibrations of its rings. It could also pro-
mote the formation of hydrogen bond between Trp indole ring and the
nearby amino acids;
2. Formation of disulfide bonds S6–S127/S30–S115 that brings the whole
C-end closer to Phe31 and Phe38 residues. This affects phenylalanine
aromatic rings vibrations;
3. Formation of SdS bonds in C-terminal that affects the conformation of
the C-terminal and, possibly, the whole lysozyme. C-terminal is more
rigid in LB crystals than in classic crystals, and in larger LB crystals than
in smaller ones. This can have an impact on the mechanical properties of
LB-grown crystals, producing more rigid and stable crystals.
172 Eugenia Pechkova et al.
6. LASER-INDUCED MICRODISSECTION
AND MICROFRAGMENTATION
Laser-induced microdissection of LB nanotemplate-facilitated protein
crystals in glycerol solution results in distinct, coherently diffracting domains
(Nicolini, Belmonte, et al., 2014; Nicolini, Bragazzi, et al., 2014). Laser-
microdissection is indeed very useful in order to obtain pieces of crystals
of very small dimensions in conjuction with X-ray nanodiffraction tech-
niques capable to overcome the very common problem of twinned, defect,
aggregated and mosaic crystals. Microdissected crystals can separatel into
smaller fragments due to effects such as cavitations at domain boundaries
and solvent interpenetration. Only crystals produced according to the LB
nanotemplate technique reveal in all four proteins being tested (lysozyme,
insulin, thaumatin, and ribonuclease) domains that are highly radiation resis-
tant, while the crystals produced by the standard hanging-drop crystallization
method do not. Actually, the very same laser exposure causes the disappear-
ance of these “classical” protein crystals during the same time frame of 40 min
needed for the laser cutting in all four proteins being tested. The micro-
diffraction of microcrystals prepared by the combination of LB and laser
technologies (Schlichting & Miao, 2012; Smith, Fischetti, & Yamamoto,
2012) proves that not only the lysozyme survives the process, as shown
recently by nanodifraction, but also all three other model proteins appear
to behave similarly well, namely, insulin, thaumatin, and ribonuclease.
The result confirms the emerging of a new biophysical technique uniquely
useful for synchrotron radiation studies based on small protein microcrystals
uniquely radiation resistant when prepared by LB nanotemplate and subse-
quently fragmented by laser.
8. IN SILICO SIMULATIONS
Crystal growth has been simulated exploiting the 2D lattice Monte
Carlo algorithm and the coarse-grained hydrophobic-polar approximation,
using monomer and tetramer (aggregate) units models. These simulation
lead to the conclusion that lysozyme tetramers LB-based crystal is expected
to be slightly accelerated when compared to its monomer-based counterpart
(Siódmiak, Gadomski, Pechkova, & Nicolini, 2006).
Previously acquired in situ GISAXS spectra (Gebhardt et al., 2010;
Pechkova & Nicolini, 2011; Pechkova et al., 2010) were analyzed using
IsGISAXS software developed by Rémi Lazzari, a tool which is dedicated
to simulation of scattering from supported nanostructures. The scattering
cross section is expressed in terms of island form factor and interference func-
tion and the specificity of the grazing-incidence geometry is stressed, in par-
ticular in the evaluation of the island form factor in the distorted-wave Born
approximation. A full account of size and possible shape distributions is
given in the decoupling approximation, where sizes and positions are not
correlated, and in the local monodisperse approximation. Two types of
island repartitions on the substrate can be implemented: disordered systems
characterized by their particle–particle pair correlation functions, and
bidimensional crystalline or paracrystalline systems of particles.
Proteins have been modeled as cylinders, LB film thickness, found with
the best fit, was fixed at 7.4 nm for thaumatin and at 6.4 nm for lysozyme,
while the wavelength was experimentally known (0.0991 nm). Critical inci-
dent angle for thaumatin and for lysozyme was computed to be 0.71 . The
delta refraction coefficients were 3.336 106 for glass and 2.19 106 for
proteins. The beta absorption coefficients were about 0 for proteins and
1.68 108 for glass. Curves have been fitted with a w2 Levenberg–
Marquardt minimization procedure, which is an iterative technique
commonly used for solving nonlinear least squares problems, with constant
standard error bars of sR/R ¼ 0.005 by means of the IsGISAXS software.
At 100 min, the particle radius of thaumatin is about 5.62 nm, while the
LB film layer thickness is about 2.25 nm, with a height ratio of 1 nm. At
900 min from the start of the experiment, the particle radius has increased
up to 40.89 nm, while on the contrary, the LB film layer thickness has
decreased down to 0 nm, with a decreased height ratio of 6.30 102 nm.
Thus, we confirmed the working hypothesis that the protein appears to trans-
fer directly from the nanobiostructured film into the drop to directly trigger
Advances in Nanocrystallography as a Proteomic Tool 177
9. BIOINFORMATICS
On the basis of a mass-scale analysis of crystal structures by mining the
PDB repository and confirming the hypothesis with a targeted ad hoc exper-
iment using thermostable thioredoxin from Alicyclobacillus acidocaldarius ver-
sus the mesophilic Escherichia coli counterpart, we have established a role of
the aqueous surroundings of a protein in its thermal stability (Pechkova,
Sartore, et al., 2007; Pechkova, Sivozhelezov, et al., 2007), and in particular
of the inner bounded water shell.
The introduction of LB film indeed affects the aqueous environment of
the protein leading to smaller numbers of water molecules.
This explanation has been recently confirmed by the water characteristics
in all model proteins (Fig. 5.1). The shape of the frequency distribution of
volumes occupied by water molecules is found to be different between
“classical” samples of different proteins, but surprisingly quite similar for
LB samples. LB film leads to the appearance of water molecules close to
the protein surface but occupying large volumes. The data suggest a
“quite Gaussian distribution” for LB and a “quite periodic distribution”
for classical as shown by the kurtosis and skewness analysis (Belmonte
et al., 2012; Pechkova, Scudieri, et al., 2012; Pechkova, Sivozhelezov,
et al., 2012).
In another study, we applied clustering algorithm and protein alignment,
showing how LB-based crystals can be compared with those obtained in
space. Proteins were downloaded from the PDB database (http://www.
rcsb.org/pdb/home/home.do), then we iteratively refined the choice
excluding proteins belonging to other taxa or radiation-damaged structures,
and finally, we subdivided the structures using the crystallization procedure
as variable (Pechkova, Bragazzi, Bozdaganyan, Belmonte, & Nicolini,
2014). The most accurate method of three-dimensional (3D) protein struc-
ture alignment algorithm based on the TM-align topological score was used,
root mean square deviation (RMSD) for C-a atoms was used as the similar-
ity measure for all structures: All the calculations were performed using the
web-based protein structure comparison tool ProCKSI.
The clustering algorithm was used selecting the Ward distance, both as
all-against-all and as all-against-target options.
178 Eugenia Pechkova et al.
We found that for lysozyme and human insulin structures were abso-
lutely comparable (Fig. 5.3, left), while similar evidence was collected also
for thaumatin and proteinase K.
From clustering algorithm, bioinformatics, and biostatistics, we can con-
clude that:
1. Bioinformatics and clustering algorithms can be applied to the study and
modeling of proteins crystallized according to different crystallization
techniques;
2. According to the clustering algorithm and statistical analysis of parame-
ters like resolution, B-factor, and solvent content, LB-based and micro-
gravity proteins are comparable and different from proteins crystallized
with other techniques.
2wrwA-1
LB-space cluster
2wrvA-1
-1
2wrxA-1
2wruA-1
A-1
2ws4A
2ws1A
2ceu
1
0A-
2ws0
1gu
A-1
1b
-1
A-1
2vk
-1
9e
jA-1
3uts
A
3v
1
7y
-1
A-
A-1
19
AA
3p
1
3e
7z
A-
A-
lin
2x
3e
8q
3k
su
1
1
A-
A-
2r in
2c
q6
b_
1
36
8r
A-
_l
2c
1l A-
1
ph 1 HN -1
A- 4I A
1h
tvA
1
3i3z -1 0.075
0A
miA-1 3brrA-1
2o 0.070
3ir0A Classical
A-1
2ws6 -1
3q6e
pA-1 A-1 LB
1w8 1os 0.069
1
11- 3A
-1
2om 1o
Space-grown
1-1 s4
A-1
o m0 2w 0.068
2 -1 s7
lzA 1 2g A-
2o - 54 1
l yA 1 2w A- 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
2o A- by 1
z9 A-
2w
Time (ps)
1
u 1
A-
1
c0
2g 2A-1
1
iu
A-
A-
2q
56
1
3z
33
1
A-
A-
1q
s
1
3p
mh
1zn
A-
1
iyA
1
1
1qiz
rA-
mg
2o
1zeg
1A-
A-1
1zehA-
jA-1
-1
2r34A-1
3rovA-1
2w44A-1
1qj0A-1
2o
3zq
A-1
3zu
1xda
A-1
1
Figure 5.3 Theoretical/semi-theoretical approaches for investigating Langmuir–Blodgett (LB)-grown crystals features and behaviors in com-
parison with other crystallization techniques. Bioinformatics analysis is shown at left, while at right, the radius of gyration simulated with
molecular dynamics (MD). LB crystal and those grown in space tend to behave in a similar way, strikingly different from the behavior of
classical crystals.
179
180 Eugenia Pechkova et al.
folding, and it was the first member of heat-shock protein 60 (hsp60) family
to be identified, being recognized as a chromosomally encoded product
whose deficiency resulted in defective morphogenesis of bacteriophage
T4 head structures and T5 tail structures (Pechkova, Tripathi, Spera, &
Nicolini, 2008). Despite a huge body of studies and investigations, the pre-
cise molecular mechanisms of substrate recognition and protein folding are
not exactly known (Azia, Unger, & Horovitz, 2012).
GroEL is a protein of high interest in the field of biosensors for gut micro-
biota sensing and has a clinical relevance, being utilized in sublingual vacci-
nation against atherosclerosis (Hagiwara et al., 2014), and against diarrhea
and colitis (Péchiné, Hennequin, Boursier, Hoys, Collignon, 2013). More-
over, GroEL is also involved in the suppression of amyloidogenesis (Yagi-
Utsumi et al., 2013) and this could have significant therapeutic implications
in the field of protein disorder and aggregation-induced disorders, also fos-
tering the discovery of new drugs and inhibitors ( Johnson et al., 2014).
It could be anticipated that LB-based crystallization of GroEL could open
new perspectives in the chaperonin biochemistry and physiopathology.
11.4. Rhodopsin
Rhodopsin has a molecular weight of 40 kDa and consists of an apoprotein,
opsin (348 amino acid residues), a chromophore, 11-cis-retinal, covalently
bound to Lys296 via a protonated Schiff base (PSB), and two oligosaccharide
chains (Shichi & Rafferty, 1980).
It crosses the membrane with seven a-helices which constitute as much
as 60% of its secondary structure and which appear oriented mostly perpen-
dicular to the plane of the disk membrane (Unger & Schertler, 1995; Unger,
Hargrave, Baldwin, & Schertler, 1997).
The rhodopsin chromophore, 11-cis-retinal, is located in a hydrophobic
pocket between the helices (Palczewski, 2012); this covalent bond of the
chromophore contributes to the tightly held rhodopsin in a nonsignaling
conformation. The extracellular domain of rhodopsin is relatively rigid,
which may help to reduce spontaneous activation of the receptor in the
absence of light (Smith, 2010).
The visual rhodopsin is a typical representative not only of the retinal-
containing proteins but also concurrently of a large family of G-protein-
coupled receptors (class A G-protein-coupled receptors or GPCRs)
(Lodowski, Angel, & Palczewski, 2009).
A better molecular understanding of rhodopsin structure could open
new perspectives in the treatment of ophthalmological diseases, such as ret-
initis pigmentosa (RP) and other disorders related to retinal degeneration
(Hollingsworth & Gross, 2012). Mutations related to RP cause protein mis-
folding and could be properly targeted by chaperones and derivative mole-
cules (Mendes, Zaccarini, & Cheetham, 2010).
Bacteriorhodopsin can be used for nanobioelectronics (Wagner, Greco,
Ranaghan, & Birge, 2013), for building and implementing 3D optical
Advances in Nanocrystallography as a Proteomic Tool 183
11.5. Globins
Globins are heme-containing proteins involved in binding, transporting,
and delivering oxygen and other nutrients. They are typically composed
of eight a-helices that fold themselves into a three-over-three a-helical
sandwich structure. In particular, Hell’s gate globin I (HGbI), a single-
domain protein with 133 residues, was identified from the genome of
Methylacidiphilum infernorum (Teh et al., 2011), an aerobic, acidophilic,
and thermophilic obligate methanotroph that grows optimally at 608 C
and pH 2.0.
HGbI is structurally homologous to mammalian neuroglobins. Its partic-
ular features are:
1. High affinity and avidity for the oxygen;
2. Negligible auto-oxidation in the pH range of 5.2–8.6 and temperature
range of 25–50 C;
3. Unique resistance to the extreme acidity and hostile environments.
These features make globin quite interesting and attractive for building
and implementing bioelectronic devices and biosensors (Chan, 2001) such
as oxygen (Perutz, Paoli, & Lesk, 1999), nitric oxide sensor (Xu, Wu, &
Zhao, 2013), useful for a vast array of applications in the field of molec-
ular nephrology (Palm, Nordquist, & Buerk, 2007) to anesthesiology
(Collison & Meyerhoff, 1990).
11.6. Insulin
Insulin is a 51-amino acid dimer protein, with a molecular weight of
5.8 kDa. The two chains are linked together by disulfide bonds. It is a clin-
ically relevant peptide hormone, produced by beta cells of the pancreas, and
plays a major role in the regulation of carbohydrate and fat metabolism in the
body. Its dysregulation causes diabetes mellitus, metabolic syndrome, and
other metabolic disorders. Insulin is required for the management of patients
with diabetes and the discovery of more active biosimilar insulins represents
a therapeutically important advancement (Heinemann, 2012). Our solved
LB-grown insulin structure has one of the best and lowest resolutions among
all the insulin crystals deposited in PDB.
184 Eugenia Pechkova et al.
12. CONCLUSIONS
LB-based crystallography has proved successful in solving the structure
of both target proteins (Fig. 5.1) and proteins difficult to crystallize with the
conventional techniques (Fig. 5.4).
LB-grown crystals have a lot of interesting features and properties: from
resistance to radiation to better domains and regular shape.
A future step of LB-based crystallography will be the structure determi-
nation of further membrane proteins (Moraes, Evans, Sanchez-Weatherby,
Newstead, & Stewart, 2014) and cytochromes (Nicolini & Pechkova, 2006;
Paternolli, Ghisellini, & Nicolini, 2007; Sivozhelezov et al., 2006; Spera
et al., 2013) that play a pivotal role in nanomedicine and above all person-
alized medicine, being the targets of commonly used drugs.
REFERENCES
Al-Hussein, M., Schindler, M., Ruderer, M. A., Perlich, J., Schwartzkopf, M., Herzog, G.,
et al. (2013). In situ X-ray study of the structural evolution of gold nano-domains by
spray deposition on thin conductive P3HT films. Langmuir, 29(8), 2490–2497.
Arya, S. K., Datta, M., & Malhotra, B. D. (2008). Recent advances in cholesterol biosensor.
Biosensors & Bioelectronics, 23(7), 1083–1100.
Azia, A., Unger, R., & Horovitz, A. (2012). What distinguishes GroEL substrates from other
Escherichia coli proteins? The FEBS Journal, 279(4), 543–550.
Bass, M., Berman, A., Singh, A., Konovalov, O., & Freger, V. (2010). Surface
structure of Nafion in vapor and liquid. The Journal of Physical Chemistry. B, 114(11),
3784–3790.
Belmonte, L., Pechkova, E., Tripathi, S., Scudieri, D., & Nicolini, C. (2012). Langmuir-
Blodgett nanotemplate and radiation resistance in protein crystals: State of the art. Critical
Reviews in Eukaryotic Gene Expression, 22(3), 219–232.
Berweger, S., Neacsu, C. C., Mao, Y., Zhou, H., Wong, S. S., & Raschke, M. B. (2009).
Optical nanocrystallography with tip-enhanced phonon Raman spectroscopy. Nature
Nanotechnology, 4(8), 496–499.
Bian, Y., Ye, M., Wang, C., Cheng, K., Song, C., Dong, M., et al. (2013). Global screening
of CK2 kinase substrates by an integrated phosphoproteomics workflow. Scientific
Reports, 3, 3460.
Bozdaganyan, M., Bragazzi, N. L., Pechkova, E., Shaytan, K., & Nicolini, C. (2014). Iden-
tification of best protein crystallization methods by molecular dynamics. Critical Reviews
in Eukaryotic Gene Expression, in press.
Bragazzi, N. L., Pechkova, E., Scudieri, D., Terencio, T. B., Adami, M., & Nicolini, C.
(2012). Recombinant laccase: II. Medical biosensor. Critical Reviews in Eukaryotic Gene
Expression, 22(3), 197–203.
Buljan, M., Radić, N., Bernstorff, S., Dražić, G., Bogdanović-Radović, I., & Holý, V.
(2012). Grazing-incidence small-angle X-ray scattering: Application to the study of
quantum dot lattices. Acta Crystallographica. Section A, 68(Pt. 1), 124–138.
Chan, M. K. (2001). Recent advances in heme-protein sensors. Current Opinion in Chemical
Biology, 5(2), 216–222.
Chayen, N. E. (2003). Protein crystallization for genomics: Throughput versus output.
Journal of Structural and Functional Genomics, 4(2–3), 115–120.
Chayen, N. E. (2004). Turning protein crystallisation from an art into a science. Current
Opinion in Structural Biology, 14(5), 577–583.
Chayen, N. E. (2007). Optimization techniques for automation and high throughput.
Methods in Molecular Biology, 363, 175–190.
186 Eugenia Pechkova et al.
Nicolini, C., Belmonte, L., Maksimov, G., Brazhe, N., & Pechkova, E. (2013). In situ mon-
itoring by raman spectroscopy of lysozyme conformation during “nanotemplate”
induced crystallization. Journal of Microbial & Biochemical Technology, 6, 009–016.
Nicolini, C., Belmonte, L., Riekel, C., Koenig, C., & Pechkova, E. (2014). Langmuir-
Blodgett nanotemplate crystallization combined to laser micro-fragmentation uniquely
characterize protein crystals by synchrotron micro-diffraction. American Journal of
Biochemistry and Biotechnology, 10(1), 22–30.
Nicolini, C., Bezerra, T., & Pechkova, E. (2012). Protein nanotechnology for the new design
and development of biocrystals and biosensors. Nanomedicine (London, England), 7(8),
1117–1120.
Nicolini, C., Bragazzi, N., & Pechkova, E. (2012). Nanoproteomics enabling personalized
nanomedicine. Advanced Drug Delivery Reviews, 64(13), 1522–1531.
Nicolini, C., Bragazzi, N., & Pechkova, E. (2013). From nanobiotechnology to organic and
biological monitoring of health and environment for biosafety. Journal of Bioanalysis and
Biomedicine, 5, 108–117.
Nicolini, C., Bragazzi, N. L., Pechkova, E., & Lazzari, R. (2014). Ab initio semi-quantitative
analysis of micro-beam grazing-incidence small-angle X-ray scattering (M-GISAXS)
during protein crystal nucleation and growth. Journal of Proteomics & Bioinformatics, 7,
064–070.
Nicolini, C., Bruzzese, D., Cambria, M. T., Bragazzi, N. L., & Pechkova, E. (2013). Recom-
binant laccase: I. Enzyme cloning and characterization. Journal of Cellular Biochemistry,
114(3), 599–605.
Nicolini, C., Bruzzese, D., Sivozhelezov, V., & Pechkova, E. (2008). Langmuir-Blodgett based
lipase nanofilms of unique structure-function relationship. Biosystems, 94(3), 228–232.
Nicolini, C., Correia, T. B., Stura, E., Larosa, C., Spera, R., & Pechkova, E. (2013). Atomic
force microscopy and anodic porous allumina of nucleic acid programmable protein
arrays. Recent Patents on Biotechnology, 7(2), 112–121.
Nicolini, C., & Pechkova, E. (2004). Nanocrystallography: An emerging technology for
structural proteomics. Expert Review of Proteomics, 1(3), 253–256.
Nicolini, C., & Pechkova, E. (2006). Nanostructured biofilms and biocrystals. Journal of
Nanoscience and Nanotechnology, 6(8), 2209–2236.
Nicolini, C., & Pechkova, E. (2010a). Nanoproteomics for nanomedicine. Nanomedicine
(London, England), 5(5), 677–682.
Nicolini, C., & Pechkova, E. (2010b). An overview of nanotechnology-based functional
proteomics for cancer and cell cycle progression. Anticancer Research, 30(6), 2073–2080.
Otálora, F., Gavira, J. A., Ng, J. D., & Garcı́a-Ruiz, J. M. (2009). Counterdiffusion methods
applied to protein crystallization. Progress in Biophysics and Molecular Biology, 101(1–3), 26–37.
Palczewski, K. (2012). Chemistry and biology of vision. The Journal of Biological Chemistry,
287(3), 1612–1619.
Palm, F., Nordquist, L., & Buerk, D. G. (2007). Nitric oxide in the kidney; direct measure-
ments of bioavailable renal nitric oxide. Advances in Experimental Medicine and Biology,
599, 117–123.
Paternolli, C., Ghisellini, P., & Nicolini, C. (2007). Nanostructuring of heme-proteins for
biodevice applications. IET Nanobiotechnology, 1(2), 22–26.
Paternolli, C., Neebe, M., Stura, E., Barbieri, F., Ghisellini, P., Hampp, N., et al. (2009).
Photoreversibility and photostability in films of octopus rhodopsin isolated from octopus
photoreceptor membranes. Journal of Biomedical Materials Research. Part A, 88(4), 947–951.
Péchiné, S., Hennequin, C., Boursier, C., Hoys, S., & Collignon, A. (2013). Immunization
using GroEL decreases Clostridium difficile intestinal colonization. PLoS One, 8(11),
e81112.
Pechkova, E., Bragazzi, N. L., Bozdaganyan, M., Belmonte, L., & Nicolini, C. (2014).
A review of the strategies for obtaining high quality crystals utilizing nanotechnologies
and space. Critical Reviews in Eukaryotic Gene Expression, in press.
Advances in Nanocrystallography as a Proteomic Tool 189
Pechkova, E., Gebhardt, R., Riekel, C., & Nicolini, C. (2010). In situ muGISAXS: I. Exper-
imental setup for submicron study of protein nucleation and growth. Biophysical Journal,
99(4), 1256–1261.
Pechkova, E., & Nicolini, C. (2002). Protein nucleation and crystallization by homologous
protein thin film template. Journal of Cellular Biochemistry, 85(2), 243–251.
Pechkova, E., & Nicolini, C. (2004). Protein nanocrystallography: A new approach to struc-
tural proteomics. Trends in Biotechnology, 22(3), 117–122.
Pechkova, E., & Nicolini, C. (2006). Structure and growth of ultrasmall protein microcrystals
by synchrotron radiation: II. microGISAX and microscopy of lysozyme. Journal of
Cellular Biochemistry, 97(3), 553–560.
Pechkova, E., & Nicolini, C. (2011). In situ study of nanotemplate-induced growth of
lysozyme microcrystals by submicrometer GISAXS. Journal of Synchrotron Radiation,
18(Pt. 2), 287–292.
Pechkova, E., & Nicolini, C. (2010). Domain organization and properties of LB lysozyme
crystals down to submicron size. Anticancer Research, 30(7), 2745–2748.
Pechkova, E., Roth, S. V., Burghammer, M., Fontani, D., Riekel, C., & Nicolini, C. (2005).
microGISAXS and protein nanotemplate crystallization: Methods and instrumentation.
Journal of Synchrotron Radiation, 12(Pt. 6), 713–716.
Pechkova, E., Sartore, M., Giacomelli, L., & Nicolini, C. (2007). Atomic force microscopy
of protein films and crystals. The Review of Scientific Instruments, 78(9), 093704.
Pechkova, E., Scudieri, D., Belmonte, L., & Nicolini, C. (2012). Oxygen-bound Hell’s gate
globin I by classical versus LB nanotemplate method. Journal of Cellular Biochemistry,
113(7), 2543–2548.
Pechkova, E., Sivozhelezov, V., Belmonte, L., & Nicolini, C. (2012). Unique water distri-
bution of Langmuir-Blodgett versus classical crystals. Journal of Structural Biology, 180(1),
57–64.
Pechkova, E., Sivozhelezov, V., & Nicolini, C. (2007). Protein thermal stability: The role of
protein structure and aqueous environment. Archives of Biochemistry and Biophysics,
466(1), 40–48.
Pechkova, E., Tripathi, S., & Nicolini, C. (2009). MicroGISAXS of Langmuir-Blodgett pro-
tein films: Effect of temperature on long-range order. Journal of Synchrotron Radiation,
16(Pt. 3), 330–335.
Pechkova, E., Tripathi, S., Ravelli, R. B., McSweeney, S., & Nicolini, C. (2009). Radiation
stability of proteinase K crystals grown by LB nanotemplate method. Journal of Structural
Biology, 168(3), 409–418.
Pechkova, E., Tripathi, S., Spera, R., & Nicolini, C. (2008). Groel crystal growth and
characterization. Biosystems, 94(3), 223–227.
Pechkova, E., Tropiano, G., Riekel, C., & Nicolini, C. (2004). Radiation stability of protein
crystals grown by nanostructured templates: Synchrotron microfocus analysis.
Spectrochimica Acta, Part B: Atomic Spectroscopy, 59(10–11), 1687–1693.
Pechkova, E., Vasile, F., Spera, R., Fiordoro, S., & Nicolini, C. (2005). Protein
nanocrystallography: Growth mechanism and atomic structure of crystals induced by
nanotemplates. Journal of Synchrotron Radiation, 12(Pt. 6), 772–778.
Pechkova, E., Zanotti, G., & Nicolini, C. (2003). Three-dimensional atomic structure of a
catalytic subunit mutant of human protein kinase CK2. Acta Crystallographica. Section D,
Biological Crystallography, 59(Pt. 12), 2133–2139.
Perutz, M. F., Paoli, M., & Lesk, A. M. (1999). Fix L, a haemoglobin that acts as an oxygen
sensor: Signalling mechanism and structural basis of its homology with PAS domains.
Chemistry & Biology, 6(11), R291–R297.
Piazza, F., Manni, S., & Semenzato, G. (2013). Novel players in multiple myeloma
pathogenesis: Role of protein kinases CK2 and GSK3. Leukemia Research, 37(2), 221–227.
Richard, M. I., Schülli, T. U., Renaud, G., Zhong, Z. Z., & Bauer, G. (2011). A combined
in situ grazing incidence small angle X-ray scattering and grazing incidence X-ray
190 Eugenia Pechkova et al.
Teh, A. H., Saito, J. A., Baharuddin, A., Tuckerman, J. R., Newhouse, J. S., Kanbe, M., et al.
(2011). Hell’s Gate globin I: An acid and thermostable bacterial hemoglobin resembling
mammalian neuroglobin. FEBS Letters, 585(20), 3250–3258.
Trembley, J. H., Wang, G., Unger, G., Slaton, J., & Ahmed, K. (2009). Protein kinase CK2
in health and disease: CK2: A key player in cancer biology. Cellular and Molecular Life
Sciences, 66(11–12), 1858–1867.
Turzhitsky, V., Qiu, L., Itzkan, I., Novikov, A. A., Kotelev, M. S., Getmanskiy, M., et al.
(2014). Spectroscopy of scattered light for the characterization of micro and nanoscale
objects in biology and medicine. Applied Spectroscopy, 68(2), 133–154.
Ubarretxena-Belandia, I., & Stokes, D. L. (2010). Present and future of membrane protein
structure determination by electron crystallography. Advances in Protein Chemistry and
Structural Biology, 81, 33–60.
Uhlmann, P., Varnik, F., Truman, P., Zikos, G., Moulin, J. F., Müller-Buschbaum, P., et al.
(2011). Microfluidic emulsion separation-simultaneous separation and sensing by mul-
tilayer nanofilm structures. Journal of Physics. Condensed Matter, 23(18), 184123.
Unger, V. M., Hargrave, P. A., Baldwin, J. M., & Schertler, G. F. (1997). Arrangement of
rhodopsin transmembrane alpha-helices. Nature, 389(6647), 203–206.
Unger, V. M., & Schertler, G. F. (1995). Low resolution structure of bovine rhodopsin deter-
mined by electron cryo-microscopy. Biophysical Journal, 68(5), 1776–1786.
Urban, J. J., Talapin, D. V., Shevchenko, E. V., & Murray, C. B. (2006). Self-assembly of
PbTe quantum dots into nanocrystal superlattices and glassy films. Journal of the American
Chemical Society, 128(10), 3248–3255.
Vergara, A., Lorber, B., Sauter, C., Giegé, R., & Zagari, A. (2005). Lessons from crystals
grown in the advanced protein crystallisation facility for conventional crystallisation
applied to structural biology. Biophysical Chemistry, 118(2–3), 102–112.
Wagner, N. L., Greco, J. A., Ranaghan, M. J., & Birge, R. R. (2013). Directed evolution of
bacteriorhodopsin for applications in bioelectronics. Journal of the Royal Society, Interface,
10(84), 20130197.
Wakayama, N. I., Yin, D. C., Harata, K., Kiyoshi, T., Fujiwara, M., & Tanimoto, Y. (2006).
Macromolecular crystallization in microgravity generated by a superconducting magnet.
Annals of the New York Academy of Sciences, 1077, 184–193.
Wickenheisser, J. K., Biegler, J. M., Nelson-Degrave, V. L., Legro, R. S., Strauss, J. F., 3rd.,
& McAllister, J. M. (2012). Cholesterol side-chain cleavage gene expression in theca
cells: Augmented transcriptional regulation and mRNA stability in polycystic ovary syn-
drome. PLoS One, 7(11), e48963.
Xu, M. Q., Wu, J. F., & Zhao, G. C. (2013). Direct electrochemistry of hemoglobin at a
graphene gold nanoparticle composite film for nitric oxide biosensing. Sensors (Basel,
Switzerland), 13(6), 7492–7504.
Yagi-Utsumi, M., Kunihara, T., Nakamura, T., Uekusa, Y., Makabe, K., Kuwajima, K.,
et al. (2013). NMR characterization of the interaction of GroEL with amyloid b as a
model ligand. FEBS Letters, 587(11), 1605–1609.
Zheng, B., Gerdts, C. J., & Ismagilov, R. F. (2005). Using nanoliter plugs in microfluidics to
facilitate and understand protein crystallization. Current Opinion in Structural Biology,
15(5), 548–555.
CHAPTER SIX
Contents
1. Introduction 194
2. The Concept of Structural Proteomics 194
3. Limited Proteolysis 195
4. Surface Modification 196
5. Hydrogen–Deuterium Exchange 200
6. Cross-linking 201
7. Additional Mass Spectrometric Techniques for the Protein Structure Analysis 207
8. Combination of Multiple Structural Proteomics Techniques 207
9. Use of Experimental Structural Proteomics Constraints in Protein Structure
Modeling 209
10. Future Directions 210
11. Conclusions 211
Acknowledgment 211
References 211
Abstract
Recent developments in the modern mass spectrometry of proteins and peptides have
resulted in significant progress in structural proteomics techniques for studying protein
structure. A variety of protein structural questions, ranging from defining protein inter-
action networks to the study of conformational changes and the structure of single pro-
teins, can be addressed using multiple mass spectrometry-based structural proteomics
approaches. Each technique provides specific structural information which can be used
as experimental structural constraints in protein structure modeling. Here, we describe
recent developments in limited proteolysis, surface modification, hydrogen–deuterium
exchange, ion mobility, and cross-linking—all combined with modern mass spectro-
metric techniques—for the studying protein structure.
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 193
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00006-3
194 Evgeniy V. Petrotchenko and Christoph H. Borchers
1. INTRODUCTION
The knowledge of protein structures is crucial for understanding of the
functioning of biological systems in healthy and disease states. The recent
revolution in mass spectrometry-based proteomics has revived an interest
in the traditional protein chemistry methods for studying protein structure.
The combination of methods such as limited proteolysis, chemical surface
modification, hydrogen–deuterium exchange (HDX), cross-linking, and
affinity labeling with mass spectrometry has led to the new field of structural
proteomics. Each of these methods provides specific and unique information
on the protein system studied. For example, limited proteolysis can indicate
portions of folded proteins that are accessible to a large probe (a proteolytic
enzyme) and must therefore be exposed to the solvent. Similarly, chemical
surface modification can provide similar information on the accessibility of a
particular amino acid residue to a smaller probe (a modification reagent).
HDX of the amide protons in the peptide bonds can provide data on the
hydrogen bonding status and accessibility, and, therefore, the presence of
secondary structure elements in the protein sequence. Chemical cross-
linkers of different lengths form a “molecular ruler” which can provide dis-
tances between the cross-linked residues (Fasold, Klappenberger, Meyer, &
Remold, 1971; Green, Reisler, & Houk, 2001; Peters & Richards, 1977).
Taken together, these methods provide a set of structural constraints on the
folded protein or protein complex being studied. In this review, we describe
the current status of these various structural proteomics methodologies, and
their application to the elucidation of the structures of proteins and protein
complexes, using the example of our recent study of prion protein conver-
sion and aggregation (Serpa, Patterson, et al., 2013).
3. LIMITED PROTEOLYSIS
The limited proteolysis method is based on short controlled exposures
of the protein to a proteolytic enzyme. The first cleavage of the protein
occurs while the tertiary and quaternary structure of the protein complex
should still be preserved, so the initial cleavage sites should be restricted to
the outermost regions of the protein subunit surfaces—that is, those are
accessible to the active site of the proteolytic enzyme. Because most enzymes
are globular proteins with molecular weights of at least 15–20 kDa, the
location of the cleavage sites will reflect their accessibility to a nearly spherical
probe, whose diameter corresponds to the size of the enzyme used.
Several mass spectrometric approaches can be used for the determination
of the cleavage sites. The most common approach is to first characterize the
196 Evgeniy V. Petrotchenko and Christoph H. Borchers
limited proteolysis reaction by SDS-PAGE. Serial time points for short (e.g.,
1–5 min) exposures of the protein to the diluted proteolytic enzyme (e.g.,
1:100 enzyme:substrate ratio) are used, and the reactions quenched and the
products are separated by SDS-PAGE. The process of proteolysis can be
visualized by time-wise appearance of the proteolytic fragments resulting
from the enzymatic cleavage. The fragments that are first to appear can then
be identified by in-gel digestion followed by peptide mapping, which will
indicate the sites of cleavage (Fig. 6.1A). Alternatively, cleavage sites can be
deduced from measuring the exact mass of the entire fragment by Orbitrap/
FTICR mass spectrometry and top-down MS/MS analysis.
Using the former approach, we have characterized conformational
changes occurring in course of native (PrPC) to aggregated state (PrPb) con-
version of prion proteins. We demonstrated increased protection of the
K110 cleavage site in PrPb compared to PrPC, suggesting intra- and/or
inter-protein interactions; and increased cleavage at sites in the region of res-
idues 149–156 in PrPb, but not in PrPC, which indicates increased exposure
of the hydrophobic residues in this region (Fig. 6.1B). These changes indi-
cate unfolding or rearrangement of the C-terminal portion of the amphi-
pathic helix 1 (H1) during the PrPC to PrPb conversion process (Serpa,
Patterson, et al., 2013).
4. SURFACE MODIFICATION
Surface modification provides information similar to that obtained
from limited proteolysis approach (i.e., protein surface accessibility), but
the determined accessibilities are to a smaller probe, in this case, a modifi-
cation reagent. The basis of this method is a chemical reaction of the protein
with a water-soluble modification reagent. Chemical modification of the
protein surface thus allows the determination of which regions of the pro-
teins are exposed to the solvent. Although the microenvironment can have a
significant influence on the reactivity of amino acid residues, it is mainly
those functional groups that are solvent exposed (i.e., which are located
on the surface of the protein molecules) which will be modified with amino
acid specific reagents. Identification of these modification sites thus indicates
which amino acid residues are on the protein surfaces and are in contact with
the solvent. The regions of the protein that are internal or are involved in
formation of interprotein contacts are shielded from the modification
reagent and consequently remain largely unmodified. Thus, analysis of
the distribution of the chemical modification sites for a multisubunit protein
A
Intact protein
Limited proteolysis
Digestion
Peptides derived from
protein cleavage products
MS analysis
10 min
20 min
30 min
10 min
30 min
10 min
20 min
30 min
0 min
1 min
5 min
0 min
1 min
5 min
0 min
1 min
5 min
0 min
1 min
5 min
98 188
62 98
49 62
38 49
undig
1 38 undig
28 2
17 28 1
14 17 3
3
14 5
6 4 2
6 4
Trypsin Pepsin
Figure 6.1 Limited proteolysis. (A) Principle of limited proteolysis cleavage-site deter-
mination by peptide mapping of the cleavage products. Following a short controlled
exposure to the proteolytic enzyme, cleavage products are separated by SDS-PAGE
and are in-gel digested. Peptide mapping of the cleavage products indicates the cleav-
age site. (B) An example of a differential limited proteolysis study of the native (PrPC)
and pathological (PrPb) states of the prion protein. Different patterns of limited prote-
olysis products were observed for the two states of the protein. Peptide mapping anal-
ysis of the cleavage products (indicated by the arrows) revealed that K110 and the
aa149–156 region are differentially accessible in the two forms of the prion protein.
Reprinted from Serpa, Patterson, et al. (2013), with permission.
198 Evgeniy V. Petrotchenko and Christoph H. Borchers
(Continued)
200 Evgeniy V. Petrotchenko and Christoph H. Borchers
5. HYDROGEN–DEUTERIUM EXCHANGE
HDX is based on the principle that protein backbone hydrogens can
be exchanged with deuterium upon exposure of a protein to a D2O-based
buffer. The exchange rates for individual peptide bond amide hydrogen
atoms are dependent on the protein’s structure: tightly hydrogen-bonded
segments undergo very slow exchange, while disordered regions exchange
much more rapidly. The hydrogen bonding of the amide hydrogen in the
amide bond of a particular amino acid residue may indicate its involvement
in secondary structure elements and/or exposure to the solvent. Short con-
trolled immersion of the protein or protein complex into a D2O solution
will lead to the replacement of the exchangeable hydrogens on the protein
surface with deuterium atoms from the solvent. Because deuterium is twice
as heavy as hydrogen, the exchange can be readily detected and quantified by
mass spectrometry. There are two general strategies to assess the location and
extent of the exchange: bottom-up and top-down analysis. In the bottom-
up approach, the protein is quickly digested, usually with pepsin under con-
ditions of low pH and low temperature at which the peptide bond amide
hydrogen exchange rate is minimal. The peptides produced are then ana-
lyzed by mass spectrometry to determine the relative amount of exchange
that has occurred. In the top-down approach, the intact protein is exposed to
time-controlled incubation in D2O buffer, and is infused into mass spec-
trometer and analyzed by MS and MS/MS.
We have developed this top-down method in combination with
electron-capture dissociation (ECD)-FTICR MS (Pan, Han, Borchers, &
Konermann, 2008, 2009). ECD is a rapid fragmentation technique that
produces selective fragmentation of peptide bonds, avoiding hydrogen scram-
bling (i.e., migration of the amide hydrogens along peptide chain), and pro-
duces an extensive series of c- and z-ions covering the protein sequence,
with most fragments differing by a single residue. By comparing the masses
of the consecutive fragments in both the c- and z-series, the exchange rate at
nearly single residue resolution can be determined (Fig. 6.3A).
We have applied this approach to the analysis of both the secondary
structures of intact proteins, as well as conformational changes, as in the case
of the prion protein conversion from PrPC to PrPb. The protein solution is
continuously mixed in a capillary—first with D2O, then with an acidic
quenching solution, and then the solution is directly infused into the mass
spectrometer. Using this approach, we determined that approximately
38 amides were protected from exchange in PrPC, while only 23 are protec-
ted in the misfolded PrPb form. In other words, 15 amides became unpro-
tected when PrP changed from the monomer to the oligomer. The region of
deprotection was localized to residues 148–164, which is the stretch of the
protein sequence encompassing H1–b2 (Fig. 6.3B). HDX deprotection in
this region indicates the loss of the secondary structure (melting of H1, dis-
assembly of the b-sheet involving the b2 strand) and/or disruption of the
H1–b2/H2–H3 interface (Serpa, Patterson, et al., 2013).
6. CROSS-LINKING
The idea behind the use of cross-linking to determine a protein’s
structure is straightforward: to introduce new covalent bonds between pairs
of functional groups in the protein in order to identify cross-linked sites
and—based on the length of the cross-linking bridges formed—to deduce
the distances between these cross-linked sites (Petrotchenko & Borchers,
2010a). These distances, in turn, can be used as constraints in the protein
structure model-building process, and/or as characteristic features of the
protein’s conformational changes.
The workflow in a typical “bottom-up” mass spectrometry-based cross-
linking experiment involves cross-linking the protein(s) of interest, optional
202 Evgeniy V. Petrotchenko and Christoph H. Borchers
information on its own, each method provides different and specific structural
information on the protein. Thus, a combination of these multiple approaches
may provide sufficient complementary information to derive the detailed
protein structure. Results from different methods verify and support each
other findings and, ultimately, provide confidence in the final result.
For the prion protein study mentioned here, we have used limited pro-
teolysis, chemical surface modification, HDX, and cross-linking as part of
our collection of the structural proteomics tools. The data from these mul-
tiple approaches are in remarkable agreement and have provided a total of
>30 residue-specific constraints, which collectively suggest that the
rearrangement of the b1–H1–b2–H2 region is the major conformational
difference between PrPC and PrPb (Fig. 6.5). A conformational change
Figure 6.5 Summary of the structural differences between PrPC and PrPb, as revealed
by multiple structural proteomics methods. The residues, which are preferentially mod-
ified or cross-linked in the native PrPC and oligomeric PrPb samples, are highlighted in
light grey and dark grey, respectively. The preferential pepsin cleavage site for PrPb is
indicated by a light grey arrow. The region of the structure which loses protection from
hydrogen–deuterium exchange in the PrPb sample is indicated by the light grey arc.
The K185–K204, K185–K220, and K204–K220 CBDPS cross-links (light grey dashed lines)
are present only in the oligomeric PrPb sample. The K185–K220 and K204–K220 cross-
links are incompatible with the native PrPC structure, which suggests a possible confor-
mational change in the PrPb aggregated form of the protein. The data from multiple
approaches collectively suggest rearrangement of the b1–H1–b2–H2 region in PrPb.
Reprinted from Serpa, Patterson, et al. (2013), with permission.
Modern Mass Spectrometry-Based Structural Proteomics 209
in the H1–b2-rigid loop region and distortion of its contact with helices 2
and 3 would create new hydrophobic patches on the surface of the molecule,
which, in turn, could be responsible for driving the aggregation process.
Through analysis of the intra- and interprotein constraint data, only one pos-
sible dimeric structure was found that satisfied all of the constraints. Thus,
this can be considered as the first experimentally determined structure for
the early conversion and aggregation events in the prion protein’s misfolding
process. This study of prion proteins has illustrated—and validated—the
utility of applying an entire arsenal of structural proteomics methods to pro-
duce a detailed and comprehensive characterization of the conformational
changes and the aggregation process. The results obtained thus far have
encouraged us to propose a similar approach to the investigation of the struc-
ture of multiple protein systems.
11. CONCLUSIONS
In summary, mass spectrometry-based structural proteomics is already
being successfully applied to many aspects of the structural analysis of pro-
teins and protein complexes, including the analysis of protein structures and
conformational changes, the determination of protein interaction interfaces,
and for elucidating the topology of multisubunit protein complexes.
Although not discussed in this review, the first examples of the application
of structural proteomics techniques to the identification of proteome-wide
protein interactions, have recently been presented (Herzog et al., 2012;
Zheng et al., 2011). With the successful integration of multiple types of
experimental data into the modeling process, we envision that the “holy
grail” of structural proteomics—the autonomous solving of protein
structures—will be achieved in the very near future.
ACKNOWLEDGMENT
This work was supported by a Genome Canada, Genome British Columbia, Technology
Development Grant.
REFERENCES
Back, J. W., Notenboom, V., de Koning, L. J., Muijsers, A. O., Sixma, T. K., de
Koster, C. G., et al. (2002). Identification of cross-linked peptides for protein interaction
studies using mass spectrometry and 18O labeling. Analytical Chemistry, 74(17),
4417–4422.
Buncherd, H., Nessen, M. A., Nouse, N., Stelder, S. K., Roseboom, W., Dekker, H. L.,
et al. (2012). Selective enrichment and identification of cross-linked peptides to study
3-D structures of protein complexes by mass spectrometry. Journal of Proteomics, 75(7),
2205–2215.
Chen, X., Chen, Y. H., & Anderson, V. E. (1999). Protein cross-links: Universal isolation
and characterization by isotopic derivatization and electrospray ionization mass spec-
trometry. Analytical Biochemistry, 273(2), 192–203.
Chen, Z. A., Jawhari, A., Fischer, L., Buchen, C., Tahir, S., Kamenski, T., et al. (2010).
Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and
mass spectrometry. EMBO Journal, 29(4), 717–726.
Chowdhury, S. M., Du, X., Tolić, N., Wu, S., Moore, R. J., Mayer, M. U., et al. (2009).
Identification of cross-linked peptides after click-based enrichment using sequential
collision-induced dissociation and electron transfer dissociation tandem mass spectrom-
etry. Analytical Chemistry, 81(13), 5524–5532.
Fasold, H., Klappenberger, J., Meyer, C., & Remold, H. (1971). Bifunctional reagents for the
crosslinking of proteins. Angewandte Chemie International Edition in English, 10(11),
795–801.
Fujii, N., Jacobsen, R. B., Wood, N. L., Schoeniger, J. S., & Guy, R. K. (2004). A novel
protein crosslinking reagent for the determination of moderate resolution protein
212 Evgeniy V. Petrotchenko and Christoph H. Borchers
Petrotchenko, E. V., Pedersen, L. C., Borchers, C. H., Tomer, K. B., & Negishi, M. (2001).
The dimerization motif of cytosolic sulfotransferases. FEBS Letters, 490(1–2), 39–43.
Petrotchenko, E. V., Serpa, J. J., Berjanskii, M., Suriyamongkol, B. P., Wishart, D. S., &
Borchers, C. H. (2012). Use of proteinase K non-specific digestion for selective and
comprehensive identification of interpeptide crosslinks: Application to prion proteins.
Molecular and Cellular Proteomics, 11(7), M111.013524.
Petrotchenko, E. V., Serpa, J. J., & Borchers, C. H. (2010). Use of a combination of isoto-
pically coded cross-linkers and isotopically coded N-terminal modification reagents for
selective identification of inter-peptide crosslinks. Analytical Chemistry, 82(3), 817–823.
Petrotchenko, E. V., Serpa, J. J., & Borchers, C. H. (2011). An isotopically-coded CID-
cleavable biotinylated crosslinker for structural proteomics. Molecular and Cellular Prote-
omics. 10(2). http://dx.doi.org/10.1074/mcp.M110.001420.
Schwieters, C. D., Kuszewski, J. J., Tjandra, N., & Clore, G. M. (2003). The Xplor-NIH
NMR molecular structure determination package. Journal of Magnetic Resonance, 160(1),
65–73.
Serpa, J. J., Parker, C. E., Petrotchenko, E. V., Han, J., Pan, J., & Borchers, C. H. (2012).
Mass spectrometry-based structural proteomics. European Journal of Mass Spectrometry,
18(2), 251–267.
Serpa, J. J., Patterson, A. P., Pan, J., Han, J., Wishart, D. S., Petrotchenko, E. V., et al. (2013).
Using multiple structural proteomics approaches for the characterization of prion pro-
teins. Journal of Proteomics, 81, 31–42.
Serpa, J. J., Petrotchenko, E. V., Wishart, D. S., & Borchers, C. H. (2013). Using
isotopically-coded hydrogen peroxide as a surface modification reagent for the structural
characterization of prion-protein aggregates. Presented at the 61st ASMS Conference on
Mass Spectrometry and Allied Topics, Minneapolis, MN.
Sohn, C. H., Agnew, H. D., Lee, J. E., Sweredoski, M. J., Graham, R. L., Smith, G. T., et al.
(2012). Designer reagents for mass spectrometry-based proteomics: Clickable cross-
linkers for elucidation of protein structures and interactions. Analytical Chemistry,
84(6), 2662–2669.
Taverner, T., Hall, N. E., O’Hair, R. A. J., & Simpson, R. J. (2002). Characterization of an
antagonist interleukin-6 dimer by stable isotope labelling, cross-linking and mass spec-
trometry. Journal of Biological Chemistry, 277(48), 46487–46492.
Wang, B., & Hakansson, K. (2008). Design and evaluation of a novel homobifunctional
cross-linker with selective metal dioxide-based enrichment potential. Presented at the
56th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO.
Yan, F., Che, F. Y., Rykunov, D., Nieves, E., Fiser, A., Weiss, L. M., et al. (2009). Non-
protein based enrichment method to analyze peptide cross-linking in protein complexes.
Analytical Chemistry, 81(17), 7149–7159.
Yang, L., Zheng, C., Weisbrod, C. R., Tang, X., Munske, G. R., Hoopmann, M. R., et al.
(2012). In vivo application of photocleavable protein interaction reporter technology.
Journal of Proteome Research, 11(2), 1027–1041.
Young, M. M., Tang, N., Hempel, J. C., Oshiro, C. M., Taylor, E. W., Kuntz, I. D., et al.
(2000). High throughput protein fold identification by using experimental constraints
derived from intramolecular cross-links and mass spectrometry. Proceedings of the National
Academy of Science USA, 97, 5802–5806.
Zheng, C., Yang, L., Hoopmann, M. R., Eng, J. K., Tang, X., Weisbrod, C. R., et al.
(2011). Cross-linking measurements of in vivo protein complex topologies. Molecular
and Cellular Proteomics, 10(10), M110.006841.
CHAPTER SEVEN
Organellar Proteomics of
Embryonic Stem Cells
Faezeh Shekari*,†, Hossein Baharvand†,{,1,
Ghasem Hosseini Salekdeh*,},1
*Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute for Stem
Cell Biology and Technology, ACECR, Tehran, Iran
†
Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran
{
Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute
for Stem Cell Biology and Technology, ACECR, Tehran, Iran
}
Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran
1
Corrresponding authors: e-mail address: [email protected]; [email protected]
Contents
1. Introduction 215
2. Organelle Proteome Analysis of ESC 219
3. Subcellular Fractionation: Current Approaches and Challenges 223
4. Organelle Proteomics Databases and Tools 225
5. Concluding Remarks 226
References 227
Abstract
Embryonic stem cells (ESCs) are undifferentiated cells with two common remarkable
features known as self-renewal and differentiation. Proteomics plays an increasingly
important role in understanding molecular mechanisms underlying self-renewal and
pluripotency of ESCs and their applications in cell therapy and developmental biology
studies. As the function of a protein is strongly associated with its localization in cell, a com-
plete and accurate picture of the proteome of ESCs cannot be achieved without knowing
the subcellular locations of proteins. Subcellular fractionation allows enrichment of low
abundant proteins and signaling complexes and reduces the complexity of the sample.
It also provided insight into tracking proteins that shuttle between different compart-
ments. Despite the substantial interest and efforts in ESC subcellular proteomics area, pro-
gress has been relatively limited. In this review, we present an overview on current status of
ESCs organelle proteomics research and discuss challenges in subcellular proteomics.
1. INTRODUCTION
Embryonic Stem Cells (ESCs) have commendable attributes including
indefinite proliferation potential accompany with preservation of the ability
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 215
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00007-5
216 Faezeh Shekari et al.
Figure 7.1 Various aspects of ESC technology (branches) involved in the function of
plenty of proteins.
Organellar Proteomics of Embryonic Stem Cells 217
heterogeneous populations into distinct classes, which can then be used for
further biological studies or cell therapy.
The first large-scale analysis of ESC PM proteome was performed using
cell surface biotinylation along with density gradient centrifugation to purify
the PM in mouse ESCs (Nunomura et al., 2005; Table 7.1). This approach
was also utilized to profile membrane proteome of human ESCs (Gu et al.,
2011) and mouse ESCs (Gu et al., 2010; Intoh et al., 2009; Wollscheid et al.,
2009). Gel electrophoresis combined with mass spectrometry approaches
were also applied for membrane proteomics analysis of mouse (Intoh
et al., 2009) and human (Gerwe et al., 2011; Mcquade et al., 2009;
Shekari et al., 2011) ESCs. Fourier transform LC–ESI–MS/MS and
MS(3) mass spectrometry has been used to evaluate membrane proteome
of human ESCs independent of culture conditions (Harkness et al.,
2008).Various sample preparation and digestion procedures were also exam-
ined to evaluate their efficiency, quality, and compatibility with subsequent
mass spectrometry analysis (Dormeyer et al., 2008). Stable isotope labeling
by amino acids in cell culture (SILAC) for human ESC lines was succeeded
in resolving more than 1000 membrane proteins (Prokhorova et al., 2009;
Sarkar et al., 2012). Furthermore, applying chemoproteomic targeting strat-
egy for discovery of cell surface N-glycoproteins of mouse ESC and induced
pluripotent stem cell (iPSC) lines, resulted in identification of 500 cell sur-
face proteins (Gundry et al., 2012). Despite of these valuable efforts, our
knowledge of membrane proteins is still very limited. Approximately
20–30% of all genes in an organism encode integral membrane proteins,
which are far beyond current available data. Analysis of membrane proteins
requires methods that solve problems such as contamination of intracellular
components, protein insolubility, low abundance, and loss of hydrophobic
peptides, which prevent protein identification.
Although ER is a runway for ribosomes to translate and/or modify
newly synthesized proteins, it has many other important functions. ER is
the main site of lipid synthesis and Ca2+ storage and membrane contact sites
involving the ER provide an important function in both of these exchange
reactions to other organelles (Helle et al., 2013). Separation of the ER from
the PM results in misregulation of phosphoinositide signaling followed by
accumulation of PI4P levels at the PM and constitutively activation of
the unfolded protein response (Manford, Stefan, Yuan, Macgurn, & Emr,
2012). The ER and mitochondria contact sites have also many roles includ-
ing coordination of calcium transfer, regulation of mitochondrial fission,
Organellar Proteomics of Embryonic Stem Cells 221
derived neural stem cell (Barthelery, Jaishankar, Salli, & Vrana, 2009) were
compared using two-dimensional difference gel electrophoresis (2DIGE).
Williamson et al. could identify some of important transcription factors
through nuclear proteomics (Williamson et al., 2008). Giving the limitation
of gel based proteomics, application of quantitative mass spectrometry pro-
teomics approaches will allow a more detailed and comprehensive insight
into nuclear proteome.
Despite the importance of mitochondria in both energy processing and
life or death decision of cell of stem cells (Chen, Hsu, & Wei, 2012; Liu et al.,
2013; Prigione & Adjaye, 2010; Prigione, Fauler, Lurz, Lehrach, & Adjaye,
2010; Rehman, 2010; Son, Jeong, Kwon, & Cho, 2013; Varum et al., 2011),
no published reports is available of ESC mitochondrial proteome.
Recently, Sarkar et al. reported proteomics of membrane, nuclear, and
cytoplasmic fractions human ESCs for membrane, nuclear, and cytoplasmic
fractions (Sarkar et al., 2012; Table 7.1).
5. CONCLUDING REMARKS
Subcellular proteomics is where cell biology crossed proteomics
( Jung, Heller, Sanchez, & Hochstrasser, 2000; Millar & Taylor, 2014).
While subcellular fractionation faces many daunting challenges, it is still
one of the pillars of proteomics and there are plenty of rooms for further
improvement. As protein localization in a cell is strongly associated with
its function, a complete and accurate picture of the proteome of cells cannot
be achieved without knowing the subcellular location(s) of proteins (Au
et al., 2007; Gatto, Vizcaino, Hermjakob, Huber, & Lilley, 2010). Subcel-
lular fractionation allows enrichment of low abundant proteins and signaling
complexes and reduces the complexity of the sample. Analyzing organelle
proteome allows also tracking proteins that shuttle between different com-
partments. Recent advances in proteomics and bioinformatics tools are
essential for the future of organelle proteomics and we anticipate that
Organellar Proteomics of Embryonic Stem Cells 227
REFERENCES
Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E. A., & Mann, M.
(2003). Proteomic characterization of the human centrosome by protein correlation pro-
filing. Nature, 426, 570–574.
Au, C. E., Bell, A. W., Gilchrist, A., Hiding, J., Nilsson, T., & Bergeron, J. J. (2007).
Organellar proteomics to create the cell map. Current Opinion in Cell Biology, 19,
376–385.
Baharvand, H., Fathi, A., Van Hoof, D., & Salekdeh, G. H. (2007). Concise review: Trends
in stem cell proteomics. Stem Cells, 25, 1888–1903.
Barthelery, M., Jaishankar, A., Salli, U., Freeman, W. M., & Vrana, K. E. (2009). 2-D DIGE
identification of differentially expressed heterogeneous nuclear ribonucleoproteins
and transcription factors during neural differentiation of human embryonic stem cells.
Proteomics. Clinical Applications, 3, 505–514.
Barthelery, M., Jaishankar, A., Salli, U., & Vrana, K. E. (2009). Reptin52 expression during
in vitro neural differentiation of human embryonic stem cells. Neuroscience Letters, 452,
47–51.
Bechard, M., & Dalton, S. (2009). Subcellular localization of glycogen synthase kinase 3beta
controls embryonic stem cell self-renewal. Molecular and Cellular Biology, 29, 2092–2104.
Bock, G., Steinlein, P., & Huber, L. A. (1997). Cell biologists sort things out: Analysis and
purification of intracellular organelles by flow cytometry. Trends in Cell Biology, 7,
499–503.
Bravo-Sagua, R., Rodriguez, A. E., Kuzmicic, J., Gutierrez, T., Lopez-Crisosto, C.,
Quiroga, C., et al. (2013). Cell death and survival through the endoplasmic
reticulum-mitochondrial axis. Current Molecular Medicine, 13, 317–329.
Chen, C. T., Hsu, S. H., & Wei, Y. H. (2012). Mitochondrial bioenergetic function and
metabolic plasticity in stem cell differentiation and cellular reprogramming. Biochimica
et Biophysica Acta, 1820, 571–576.
Dormeyer, W., Van Hoof, D., Mummery, C. L., Krijgsveld, J., & Heck, A. J. (2008).
A practical guide for the identification of membrane and plasma membrane proteins
in human embryonic stem cells and human embryonal carcinoma cells. Proteomics, 8,
4036–4053.
Dreger, M. (2003). Subcellular proteomics. Mass Spectrometry Reviews, 22, 27–56.
Dunkley, T. P., Watson, R., Griffin, J. L., Dupree, P., & Lilley, K. S. (2004). Localization of
organelle proteins by isotope tagging (LOPIT). Molecular & Cellular Proteomics, 3,
1128–1134.
Fleischer, S., & Kervina, M. (1974). Subcellular fractionation of rat liver. Methods in Enzy-
mology, 31, 6–41.
Foster, L. J., De Hoog, C. L., Zhang, Y., Zhang, Y., Xie, X., Mootha, V. K., et al. (2006).
A mammalian organelle map by protein correlation profiling. Cell, 125, 187–199.
Gatto, L., Vizcaino, J. A., Hermjakob, H., Huber, W., & Lilley, K. S. (2010). Organelle pro-
teomics experimental designs and analysis. Proteomics, 10, 3957–3969.
Gauthier, D. J., Sobota, J. A., Ferraro, F., Mains, R. E., & Lazure, C. (2008). Flow
cytometry-assisted purification and proteomic analysis of the corticotropes dense-core
secretory granules. Proteomics, 8, 3848–3861.
Gerwe, B. A., Angel, P. M., West, F. D., Hasneen, K., Young, A., Orlando, R., et al. (2011).
Membrane proteomic signatures of karyotypically normal and abnormal human embry-
onic stem cell lines and derivatives. Proteomics, 11, 2515–2527.
228 Faezeh Shekari et al.
Gu, B., Zhang, J., Wang, W., Mo, L., Zhou, Y., Chen, L., et al. (2010). Global expression of
cell surface proteins in embryonic stem cells. PLoS One, 5, e15795.
Gu, B., Zhang, J., Wu, Y., Zhang, X., Tan, Z., Lin, Y., et al. (2011). Proteomic analyses
reveal common promiscuous patterns of cell surface proteins on human embryonic stem
cells and sperms. PLoS One, 6, e19386.
Gundry, R. L., Riordon, D. R., Tarasova, Y., Chuppa, S., Bhattacharya, S., Juhasz, O., et al.
(2012). A cell surfaceome map for immunophenotyping and sorting pluripotent stem
cells. Molecular & Cellular Proteomics, 11, 303–316.
Guo, T., Hua, S., Ji, X., & Sun, Z. (2004). DBSubLoc: Database of protein subcellular local-
ization. Nucleic Acids Research, 32, D122–D124.
Harkness, L., Christiansen, H., Nehlin, J., Barington, T., Andersen, J. S., & Kassem, M.
(2008). Identification of a membrane proteomic signature for human embryonic stem
cells independent of culture conditions. Stem Cell Research, 1, 219–227.
Harrison, P. M., Kumar, A., Lang, N., Snyder, M., & Gerstein, M. (2002). A question of size:
The eukaryotic proteome and the problems in defining it. Nucleic Acids Research, 30,
1083–1090.
Helle, S. C., Kanfer, G., Kolar, K., Lang, A., Michel, A. H., & Kornmann, B. (2013). Orga-
nization and function of membrane contact sites. Biochimica et Biophysica Acta, 1833,
2526–2541.
Holter, H., Ottesen, M., & Weber, R. (1953). Separation of cytoplasmic particles by cen-
trifugation in a density-gradient. Experientia, 9, 346–348.
Hornig-Do, H. T., Gunther, G., Bust, M., Lehnartz, P., Bosio, A., & Wiesner, R. J. (2009).
Isolation of functional pure mitochondria by superparamagnetic microbeads. Analytical
Biochemistry, 389, 1–5.
Intoh, A., Kurisaki, A., Yamanaka, Y., Hirano, H., Fukuda, H., Sugino, H., et al. (2009).
Proteomic analysis of membrane proteins expressed specifically in pluripotent murine
embryonic stem cells. Proteomics, 9, 126–137.
Jaishankar, A., Barthelery, M., Freeman, W. M., Salli, U., Ritty, T. M., & Vrana, K. E.
(2009). Human embryonic and mesenchymal stem cells express different nuclear
proteomes. Stem Cells and Development, 18, 793–802.
Jung, E., Heller, M., Sanchez, J. C., & Hochstrasser, D. F. (2000). Proteomics meets cell
biology: The establishment of subcellular proteomes. Electrophoresis, 21, 3369–3377.
Kausch, A. P., Owen, T. P., Jr., Narayanswami, S., & Bruce, B. D. (1999). Organelle iso-
lation by magnetic immunoabsorption. Biotechniques, 26, 336–343.
Kim, H., Wu, J., Ye, S., Tai, C. I., Zhou, X., Yan, H., et al. (2013). Modulation of
beta-catenin function maintains mouse epiblast stem cell and human embryonic stem cell
self-renewal. Nature Communications, 4, 2403.
Lawson, E. L., Clifton, J. G., Huang, F., Li, X., Hixson, D. C., & Josic, D. (2006). Use of
magnetic beads with immobilized monoclonal antibodies for isolation of highly pure
plasma membranes. Electrophoresis, 27, 2747–2758.
Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G., Levine, S. S., Kumar, R. M., et al.
(2006). Control of developmental regulators by Polycomb in human embryonic stem
cells. Cell, 125, 301–313.
Lee, Y. H., Tan, H. T., & Chung, M. C. (2010). Subcellular fractionation methods and strat-
egies for proteomics. Proteomics, 10, 3935–3956.
Liu, W., Long, Q., Chen, K., Li, S., Xiang, G., Chen, S., et al. (2013). Mitochondrial metab-
olism transition cooperates with nuclear reprogramming during induced pluripotent
stem cell generation. Biochemical and Biophysical Research Communications, 431, 767–771.
Manford, A. G., Stefan, C. J., Yuan, H. L., Macgurn, J. A., & Emr, S. D. (2012). ER-
to-plasma membrane tethering proteins regulate cell signaling and ER morphology.
Developmental Cell, 23, 1129–1140.
Organellar Proteomics of Embryonic Stem Cells 229
Marchi, S., Patergnani, S., & Pinton, P. (2014). The endoplasmic reticulum-mitochondria con-
nection: one touch, multiple functions. Biochimica et Biophysica Acta, 1837(4), 461–469.
Mcquade, L. R., Schmidt, U., Pascovici, D., Stojanov, T., & Baker, M. S. (2009). Improved
membrane proteomics coverage of human embryonic stem cells by peptide IPG-IEF.
Journal of Proteome Research, 8, 5642–5649.
Merrill, B. J. (2012). Wnt pathway regulation of embryonic stem cell self-renewal. Cold
Spring Harbor Perspectives in Biology, 4, a007971.
Millar, A. H., & Taylor, N. L. (2014). Subcellular proteomics-where cell biology meets pro-
tein chemistry. Frontiers in Plant Science, 5, 55.
Moschallski, M., Hausmann, M., Posch, A., Paulus, A., Kunz, N., Duong, T. T., et al.
(2010). MicroPrep: Chip-based dielectrophoretic purification of mitochondria.
Electrophoresis, 31, 2655–2663.
Murayama, K., Fujimura, T., Morita, M., & Shindo, N. (2001). One-step subcellular frac-
tionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-
thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main
fraction of organelles. Electrophoresis, 22, 2872–2880.
Nasrabadi, D., Larijani, M. R., Fathi, A., Gourabi, H., Dizaj, A. V., Baharvand, H., et al.
(2010). Nuclear proteome analysis of monkey embryonic stem cells during differentia-
tion. Stem Cell Reviews, 6, 50–61.
Nobelprize.org (2013). Nobelprize.org. Nobel Media AB 2013. Available from: http://www.
nobelprize.org/nobel_prizes/medicine/laureates/1974/presentation-speech.html, Accessed
8 Jan 2014 [Online].
Nunomura, K., Nagano, K., Itagaki, C., Taoka, M., Okamura, N., Yamauchi, Y., et al.
(2005). Cell surface labeling and mass spectrometry reveal diversity of cell surface markers
and signaling molecules expressed in undifferentiated mouse embryonic stem cells. Molec-
ular & Cellular Proteomics, 4, 1968–1976.
Okado-Matsumoto, A., & Fridovich, I. (2001). Subcellular distribution of superoxide dis-
mutases (SOD) in rat liver: Cu, Zn-SOD in mitochondria. The Journal of Biological Chem-
istry, 276, 38388–38393.
Pflugradt, R., Schmidt, U., Landenberger, B., Sanger, T., & Lutz-Bonengel, S. (2011).
A novel and effective separation method for single mitochondria analysis.
Mitochondrion, 11, 308–314.
Pierleoni, A., Martelli, P. L., Fariselli, P., & Casadio, R. (2007). eSLDB: Eukaryotic subcel-
lular localization database. Nucleic Acids Research, 35, D208–D212.
Prigione, A., & Adjaye, J. (2010). Modulation of mitochondrial biogenesis and bioenergetic
metabolism upon in vitro and in vivo differentiation of human ES and iPS cells. The Inter-
national Journal of Developmental Biology, 54, 1729–1741.
Prigione, A., Fauler, B., Lurz, R., Lehrach, H., & Adjaye, J. (2010). The senescence-related
mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem
cells. Stem Cells, 28, 721–733.
Prokhorova, T. A., Rigbolt, K. T., Johansen, P. T., Henningsen, J., Kratchmarova, I.,
Kassem, M., et al. (2009). Stable isotope labeling by amino acids in cell culture
(SILAC) and quantitative comparison of the membrane proteomes of self-renewing
and differentiating human embryonic stem cells. Molecular & Cellular Proteomics, 8,
959–970.
Rastogi, S., & Rost, B. (2011). LocDB: Experimental annotations of localization for Homo
sapiens and Arabidopsis thaliana. Nucleic Acids Research, 39, D230–D234.
Rehman, J. (2010). Empowering self-renewal and differentiation: The role of mitochondria
in stem cells. Journal of Molecular Medicine, 88, 981–986.
Reiland, S., Salekdeh, G. H., & Krijgsveld, J. (2011). Defining pluripotent stem cells through
quantitative proteomic analysis. Expert Review of Proteomics, 8, 29–42.
230 Faezeh Shekari et al.
Rubin, L. L., & Haston, K. M. (2011). Stem cell biology and drug discovery. BMC Biology,
9, 42.
Sarkar, P., Collier, T. S., Randall, S. M., Muddiman, D. C., & Rao, B. M. (2012). The sub-
cellular proteome of undifferentiated human embryonic stem cells. Proteomics, 12,
421–430.
Satori, C. P., Kostal, V., & Arriaga, E. A. (2012). Review on recent advances in the analysis of
isolated organelles. Analytica Chimica Acta, 753, 8–18.
Sengelov, H., & Borregaard, N. (1999). Free-flow electrophoresis in subcellular fractionation
of human neutrophils. Journal of Immunological Methods, 232, 145–152.
Shekari, F., Taei, A., Pan, T. L., Wang, P. W., Baharvand, H., & Salekdeh, G. H. (2011).
Identification of cytoplasmic and membrane-associated complexes in human embryonic
stem cells using blue native PAGE. Molecular BioSystems, 7, 2688–2701.
Sokol, S. Y. (2011). Maintaining embryonic stem cell pluripotency with Wnt signaling.
Development, 138, 4341–4350.
Son, M. J., Jeong, B. R., Kwon, Y., & Cho, Y. S. (2013). Interference with the mitochon-
drial bioenergetics fuels reprogramming to pluripotency via facilitation of the glycolytic
transition. The International Journal of Biochemistry & Cell Biology, 45, 2512–2518.
Sprenger, J., Lynn Fink, J., Karunaratne, S., Hanson, K., Hamilton, N. A., & Teasdale, R. D.
(2008). LOCATE: A mammalian protein subcellular localization database. Nucleic Acids
Research, 36, D230–D233.
Varum, S., Rodrigues, A. S., Moura, M. B., Momcilovic, O., Easley, C. A. T., Ramalho-
Santos, J., et al. (2011). Energy metabolism in human pluripotent stem cells and their
differentiated counterparts. PLoS One, 6, e20914.
Weber, P. J. A., Weber, G., & Eckerskorn, C. (2004). Isolation of organelles and
prefractionation of protein extracts using free-flow electrophoresis. Current Protocols in
Protein Science, 32, 22.5.1–22.5.21.
Williamson, A. J., Smith, D. L., Blinco, D., Unwin, R. D., Pearson, S., Wilson, C., et al.
(2008). Quantitative proteomics analysis demonstrates post-transcriptional regulation
of embryonic stem cell differentiation to hematopoiesis. Molecular & Cellular Proteomics,
7, 459–472.
Wollscheid, B., Bausch-Fluck, D., Henderson, C., O’Brien, R., Bibel, M., Schiess, R., et al.
(2009). Mass-spectrometric identification and relative quantification of N-linked cell
surface glycoproteins. Nature Biotechnology, 27, 378–386.
Yan, W., Aebersold, R., & Raines, E. W. (2009). Evolution of organelle-associated protein
profiling. Journal of Proteomics, 72, 4–11.
CHAPTER EIGHT
Contents
1. Introduction 232
2. Protein Arrays 237
2.1 Recent achievements of the protein arrays and their application
to address the study of the human proteome 246
2.2 Advantages and limitations of protein arrays 250
3. Protein-Binding DNA Arrays and Their Application to Address
the Study of DNA-Binding Proteins 254
3.1 Recent achievements of protein-binding DNA arrays and their application
to address the study of the human proteome 257
3.2 Advantages and limitations of protein-binding DNA arrays 259
4. Databases and Web Resources for PPIs and for PDIs 260
5. Conclusions and Future Perspectives 269
Acknowledgments 270
References 271
Abstract
In this report, we focus on two different array-based technologies that enable large-
scale screening of protein interactions. First, protein arrays focus on the identification
of protein–protein interactions (PPIs). Second, DNA arrays have also evolved to explore
the identification of protein–DNA interactions (PDIs), offering novel tools to control key
biological processes. Such a tool is termed protein-binding DNA arrays (also protein–
DNA arrays or protein-binding microarrays). These two array-based technologies share
unrivaled screening capabilities and constitute valid approaches to address biological
questions at the molecular level and, eventually, may be used in biomedical applica-
tions. Outstanding achievements of these technologies and their eventual application
in biomedicine are discussed here, including the identification and characterization of
Advances in Protein Chemistry and Structural Biology, Volume 95 # 2014 Elsevier Inc. 231
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00008-7
232 Juan Casado-Vela et al.
1. INTRODUCTION
The pool of molecules concomitantly contributing to regulate and
keep homeostasis in living organisms includes: nucleic acids, proteins
(including enzymes and peptides), and metabolites. All of them play essential
roles and, thus, understanding their interaction is a fundamental step that
may foster biomedical applications. Currently, there is no single technique
able to cope with this complex mixture of molecules at the same time. For
that reason, different analytical techniques combined with enrichment or
purification protocols is required to address their identification, modulation,
and to measure their dynamic changes (Fig. 8.1).
The analytical techniques used for the characterization of each molecule
are very different and the interpretation of data typically requires specializa-
tion and expertise. Thus, four areas of science emerged, termed genomics,
transcriptomics, metabolomics and proteomics. These areas share a common
basic aim, which is the large-scale identification and characterization of pools
of biological molecules. From a general and simplistic perspective, genomics
and transcriptomics address the study of the DNA and RNA molecules,
metabolomics focuses on the study of “small molecules”, differing in
chemical composition and properties. Finally, proteomics addresses the
study of proteins, enzymes and peptides. As displayed in Fig. 8.1, the
complexity of each -omic approach is significantly higher (genomics
<metabolomics < proteomics), due to the increasing repertoire of different
molecules with high complexity to be analyzed.
From all the possible interactions among biomolecules within cells, in
this report we focus on protein interactions (specifically, protein–protein
interactions (PPIs), and protein–DNA interactions (PDIs)) and relevant
techniques enabling their characterization in high-throughput format. Pro-
teins carry out the majority the biochemical reactions within cells and may
also function as signal messengers or as gene transcription factors. Thus, pro-
teins constitute frequently targets for drug design and might also serve as bio-
markers in biomedical applications. Importantly, living organisms include a
Screening of Protein–Protein and Protein–DNA Interactions 233
Figure 8.1 (A) Overview of the main types of compounds typically studied, well-
established enrichment protocols, and main techniques used. (B) A comprehensive view
of living organisms requires the functional integration of data comprising three closely
related branches of study, genomics/transcriptomics, proteomics/enzymology/
peptidomics, and metabolomics. Abbreviations: (q)PCR, quantitative polymerase chain
reaction; RT-PCR, real-time polymerase chain reaction; SNPs, single nucleotide polymor-
phism; Chip, chromatin immunoprecipitation; NMR, nuclear magnetic resonance; LC–
MS/MS, liquid chromatography coupled to tandem mass spectrometry; GC–MS, gas
chromatography coupled to tandem mass spectrometry, 2D-PAGE, two dimensional
polyacrylamide gel electrophoresis; Y2H, yeast two hybrid; TAP, tandem affinity
purification.
unraveling the identity and the dynamic changes of such protein interactions
becomes crucial since they may provide the basis for protein regulation and,
thus, tools for controlling physiological alterations and signaling networks
where proteins are involved in.
Nowadays, there are several technologies available to address the study
PPIs. Yeast two-hybrid assay (Y2H) is a well-established methodology that
permits the rapid identification of binary interactions between a chosen test
protein, termed “bait” and an interacting protein(s), termed prey(s) (Fields &
Song, 1989). Y2H methods were reviewed by Bruckner, Polge, Lentze,
Auerbach, and Schlattner (2009). Basically, the identification of interacting
proteins by Y2H is achieved by expressing the bait protein as a hybrid, fused
to the DNA-binding domain of a transcription factor, and screening it
against a library of prey candidates that are fused to the corresponding tran-
scriptional activation domain. Both fusions are expressed in yeast cells that
carry a reporter gene whose expression is under the control of the transcrip-
tion factor, such that the interaction of the “two hybrids” leads to expression
of the reporter. Other methods used to decipher PPIs include affinity
purification methods that are based on antibodies mediated enrichment
and tandem affinity purifications (TAP; see, Xu et al., 2010 for review)
which is also being widely used for protein complex purification. These
techniques, in combination with mass spectrometry have led to valuable
information about protein complexes partners and PPI. Lately, array based
methods as nucleic acid programmable protein array (NAPPA; Sibani &
LaBaer, 2011) or protein in situ arrays (PISA; He, Stoevesandt, & Taussig,
2008) became popular approaches to test interactions in high-throughput
format. Despite of robustness and development of these approaches, it has
to be noted that none of the methods used to the study of PPIs are compa-
rably better than other and typically lead to the identification of different
subsets of interactions (Braun et al., 2009; Chen et al., 2004; Chen,
Rajagopala, Stellberger, & Uetz, 2010; Chen, Zhou, Sanders, Nolan, &
Cai, 2009). Analysis of protein complexes using affinity purification
followed by mass spectrometry typically identifies directly and indirectly
associated proteins, whereas Y2H analyses identify direct, binary PPIs
(Braun et al., 2009). Moreover, different Y2H systems can be used, which
also leads to detection of markedly different subsets of interacting proteins,
reviewed in (Chen et al., 2010). Therefore, the results derived from different
PPI analyses are complementary.
Previous publications inferred different estimations of the human inter-
actome (Table 8.1) with figures widely differing from 13,000 to
Screening of Protein–Protein and Protein–DNA Interactions 235
understand protein function. In this sense, array technologies open the pos-
sibility to address this task and allow the identification of both PPIs and PDIs,
which constitute an “always-pursued” objective in science. Therefore, array
technology opens the door for addressing a plethora of biological questions
through sensitive and high-throughput (i.e., several thousand proteins/
molecules can be screened in a single experiment) approaches. The rapid
development and miniaturization of arrays led to remarkable achievements
in biomedicine. Miniaturization seems a must, in our race towards the iden-
tification of novel protein biomarkers.
2. PROTEIN ARRAYS
Protein arrays may be concisely defined as collections of proteins
attached to known positions on solid surfaces. Albeit the apparent simplicity
of the concept “protein array,” different terms and definitions may be found
in the literature such as forward-phase or reverse-phase protein arrays, tissue
or antibody protein arrays and cell-free or cell-based protein arrays
(LaBaer & Ramachandran, 2005; Lee, Magee, Gaster, LaBaer, & Wang,
2013; Liotta et al., 2003; Matarraz, Gonzalez-Gonzalez, Jara, Orfao, &
Fuentes, 2011). Importantly, different criteria may be used to classify protein
arrays, such as the type and source of the proteins printed on the arrays, their
applications or the technologies used to build and detect interactions
(Casado-Vela, Gonzalez-Gonzalez, et al., 2013). A list of types of protein
arrays described in the literature, concise description, main applications,
and references is included in Table 8.2.
Since the introduction of the first arrays published in 2000–2001 (Haab,
Dunham, & Brown, 2001; MacBeath & Schreiber, 2000; Miller, Butler,
Teh, & Haab, 2001) both printing and detection technologies evolved rap-
idly, mainly because of their potential applications. Originally, the idea of
building protein arrays was based on the immobilization of purified target
proteins (obtained by heterologous cell-based systems or using recombinant
protein technology) directly onto defined positions on the array surface.
This idea may be extended to the attachment of known sets of antibodies
to build antibody arrays (Fig. 8.2).
Figure 8.2A shows an overview of the general workflow and key steps
involved in protein array and antibody array analysis. In protein arrays, the
target proteins are attached on defined positions of the array surfaces and
they are covered with a solution of the query protein of interest. Conversely,
in antibody arrays a set of known antibodies are printed on defined positions
238 Juan Casado-Vela et al.
of the array, enabling the binding of cognate protein partners. Although pro-
tein arrays and antibody arrays are conceptually different, they both rely on
the detection and quantification of fluorescent signals on defined specific
areas of arrays. Thus, protein arrays and antibody arrays share the technol-
ogies used for digital image acquisition and image analysis, but differ in the
algorithms used for data interpretation. In the case of PPIs, the identification
of protein-binding partners is typically calculated using the z-score value—
see Dı́ez et al. (2012) for detailed description on the calculation of this and
other statistical strategies used in microarray data analysis. In antibody arrays,
two different conditions (such as control versus treatment) are typically
A B
32 blocks
4 rows ´ 4 columns
Protein arrays Antibody arrays 4 blocks 512 spots
6 rows ´ 20 columns
480 spots
48 blocks
Antibody-based fluorescent Fluorescent detection of antibody 48 blocks 25 rows ´ 32 columns
detection of binary interactions protein interactions 22 rows ´ 22 columns 38400 spots
23232 spots
Figure 8.2 (A) Outline of the general experimental workflow for protein array and antibody arrays analysis. (B) Overview of different array
configurations showing increasing number of features/spots printed and detected as round fluorescent spot signals. Low-density arrays (less
than 1000 spots) and high-density arrays (typically over 1000 spots) are shown. The highest density array (bottom-right corner) corresponds
to HuProt™ arrays, bearing more than 17,000 full length human proteins distributed in 38,400 spots printed on a single array surface the
image was provided by Dr. Ronny Schmidt from Cambridge Protein Arrays Ltd.
Screening of Protein–Protein and Protein–DNA Interactions 243
Ribosome
a a a a
b b c b b c
Differential protein
e e
ab c ab c b
B O
HRP
d a d a
(a) Positive control
(b) Printing buffer Horseradish Protein tag used for
OH OH HRP Tag
OH peroxidase purification
(c) BSA
(d) Negative control Antibody Query protein
Tyramide Activated Query protein (e) GST
Tyrosine
tyramide modification
Interacting
protein
246 Juan Casado-Vela et al.
Figure 8.3 (A) Fluorescence image protein array. Those spots displaying higher lumi-
nescence correspond to binary protein–protein interactions. A zoomed view on a single
spot displays a schematic view of the main processes taking place during de novo pro-
tein synthesis before incubation of the protein of interest (de novo protein synthesis,
antibody-based protein binding to known positions on the array). (B) Schematic view
of fluorescent (Cy3) tyramide activation by horseradish peroxidase (right panel displays
fluorescent signal amplification in those array positions where the query protein binds
to interacting proteins de novo synthesized on the array surface). Fluorescent signal
amplification only takes place if binary protein–protein interactions take place. (C) Dif-
ferential display analysis corresponding to the identification of protein–protein interac-
tions detected after the comparison of two fluorescent images. The image on the left
corresponds to NAPPA arrays incubated with a tagged protein. The image on the right
corresponds to a control experiment using only the tag. Close-up views of the digital
images clearly show a number of protein partners (printed in duplicates). Quality con-
trols including fluorescent dyes, printing buffer, BSA, GST, and negative controls are also
displayed.
Screening of Protein–Protein and Protein–DNA Interactions 247
and open new avenues for the development of protein arrays that can serve as
reliable diagnostic tests in clinical practice.
Table 8.3 Advantages and limitations of protein arrays to address the study
of protein–protein interactions
Advantages
Large-scale monitoring experiments (several thousand proteins can be monitored
in a single experiment).
Wide range of applications: screening, differential profiling, detection of
biomarkers, functional characterization of proteins, protein–protein/peptide/
molecule interactions.
Low sample consumption (especially relevant for clinical applications).
Fast experiments, allowing detection/quantitation/characterization of up to
thousands of proteins in a single experiment.
Experimental conditions can be easily controlled.
Increased protein detection levels compared to other high-throughput proteomic
technologies (e.g., LC–MS/MS).
A range of different chemistries may be used to modify solid surface (typically
glass) to achieve protein binding.
Customizable, they can be constructed “ad hoc” for specific purposes.
Proprietary antibodies raised in-house may be printed.
Disadvantages
Typically focused on binary protein–protein interactions (not optimized for the
identification of protein complexes).
Increased possibility of detecting false positives as potential interactors.
Validation experiments (coimmunoprecipitation, Western-blot, colocalization,
etc.,) are required.
The posttranslational modification status of the proteins attached on arrays is
difficult to control.
The largest “whole proteome” currently reported corresponds to a relatively
simple model organism (yeast), complete eukaryotic proteomes remain unknown.
Lack of ability to measure binding/dissociation constants.
The concentration of the target protein/molecule in the solution cannot be
measured.
The highest density arrays reported to date include 23,000 different positions
on a single array.
Several variables may alter the functionality of the proteins during the production
and/or storage of the protein arrays.
Complexity of experimental design, lack of standardized protocols.
The proteins arrayed may not be properly folded, fully functional, or optimally
oriented after binding to solid surfaces.
Deep characterization of the specificity and stability of the antibodies used in
antibody arrays is required.
252 Juan Casado-Vela et al.
list of proteins able to bind DNA and the elucidation of the specific
sequences within DNA that are recognized constitute two main objectives.
Of particular interest, is the case of transcription factors (TFs), responsible for
the transcriptional regulation through recognition and binding to specific
DNA sequences located inside regulatory domains. The elucidation of
DNA-motifs recognized by TFs is a main task for elucidating the so called
“transcriptional regulatory code” (Harbison et al., 2004). Chromatin immu-
noprecipitation (ChIP) of TF–DNA complexes and analysis of DNA
fragments with oligonucleotide arrays (ChIP-chip) or deep-sequencing
(ChIP-seq) provides an in vivo landscape of the target genes of TFs—
reviewed in Park (2009). However, ChIP-based methodologies make the
study of TF-binding sites at large scale an unaffordable task, given the neces-
sity of specific antibodies against the TFs under study. Alternatively,
PDI-arrays offer a valuable alternative to analyze and detect specific DNA
sequences-binding proteins.
Figure 8.4 shows a schematic overview of protein-binding DNA arrays
and their application to unravel PDIs. With this approach, single-stranded
DNA molecules (ssDNA) are converted into double-stranded DNAs
(dsDNA) in a primer extension reaction using universal primers comple-
mentary to all the DNA sequences. In this manner, known dsDNA
sequences remain attached on known positions of the array. Such array of
molecules can be directly incubated with soluble preparations of the protein
of interest (purified or bacterial protein extracts may be used). Finally, PDIs
are immunologically probed with Cy5 fluorescent-labeled specific anti-
bodies raised against protein epitopes (or against epitope tags).
Fluorescent-labeled primary or secondary antibodies may be used to detect
the interactions. Subsequently, specific sequences within DNA that bind
query proteins are identified after image processing using dedicated software.
Several fluorochrome-based detection strategies may be used. For
instance, Warren et al. (2006) conjugated Cy3 at a particular cysteine residue
on an unstructured portion of the protein Exd that did not affect its
functionality. More versatile and easier are those strategies based on immu-
nological detection of DNA-transcription factor complexes with
fluorochrome-conjugated antibodies. In this case, PDI-array is incubated
with the transcription factor of interest fused to an epitope tag (e.g., gluta-
thione S-transferase, GST), and bound PDI-array incubated with a labeled
antibody (Mukherjee et al., 2004). Alternatively, the protein may be fused to
a fluorescent protein (e.g., DsRed), and fluorescence directly detected
without need for multistep incubations (Kim, Lee, et al., 2009).
256 Juan Casado-Vela et al.
A B C
Synthesis of protein binding Incubation of protein of Dectection of protein-DNA
DNA arrays interest with dsDNA interactions
Cy5
DNA-binding
Protein–DNA complexes,
protein
washing for detection of
interactions
Advantages
Identification of specific DNA sequences recongized by DNA-binding
proteins in vitro.
Relatively simple methodology.
Fast: the entire process takes two working days.
No need of purification and/or labeling of the protein.
Suitable for any commercially available antibody.
Pararallelizable when using multiplexed arrays: several different proteins
can be assayed at once.
Relatively cheap with multiplexed arrays.
Data obtained in PDI-arrays correlate well with data obtained in vivo.
Aplicable to known heterodimers.
Disadvantages
In vitro system that do not yield actual targets in vivo.
Restricted to short sequences, between 8 and 11 bp. Bipartite motifs are rarely
identified.
Depends on the solubility and correct folding of the protein expressed in E. coli.
Not suitable for proteins requiring post-translational modifications for
DNA-binding.
Not suitable for proteins requiring unknown specific heterodimerizations.
previous experiments and to compare with novel experimental data. For that
reason, we also include here a directory of valuable resources (Table 8.5)
including the list, description, and links gathering information of PPIs
and PDIs.
An issue for consideration is that many databases include data derived
from prediction algorithms and computational methods. In this regard, a
number of algorithms and computational methods currently coexist and
can be used to infer the occurrence of PPIs (Gomez, Choi, & Wu, 2008;
Gong et al., 2008; Jessulat et al., 2011; Mishra, 2012; Pitre et al., 2008;
Skrabanek, Saini, Bader, & Enright, 2008). These algorithms rely on one
or more features—such as genomic sequence, topological genomic cluster-
ing, protein sequence, protein structure, protein functional/structural
domains, or evolutionary relationship—and may also take advantage of
known PPI datasets to test, train, and improve the quality of their predic-
tions. A comparative overview of prediction algorithms is beyond the scope
of this report, but it is important to underline that computational algorithms
frequently take advantage of reliable training datasets (i.e., bona-fide list of
protein interactions) to test and to improve their predictions.
More importantly, the list of candidate protein partners retrieved may
significantly differ among databases, due to the fact that the amount and
quality of the information deposited in each database are not really compa-
rable (Klingstrom & Plewczynski, 2011, reviewed PPI databases and their
sources of information). The overall information overlap among databases
is limited and, thus, gathering information from as many databases as possible
may represent an advantage if thorough information on the interactome of a
specific protein is the objective. This task currently constitutes an obstacle
that may be prohibitive in terms of time.
The reasons above justify the current trend towards the development of
web-based search engines able to gather protein interactors from multiple
databases at the same time and yielding updated information. Examples of
such engines include PSICQUIC (Aranda et al., 2011), DASMI
(Blankenburg et al., 2009), and BIPS (Garcia-Garcia, Schleker, Klein-
Seetharaman, & Oliva, 2012), recently developed and publicly available
for the scientific community. These web tools significantly simplify the
screening of information.
Nevertheless, relevant information affecting protein interactions (such
as the specific physical and biochemical parameters affecting to those
interactions) is frequently overlooked (Schleker et al., 2012) and, more
importantly, still suffers from high rates of false positives and errors
Table 8.5 Name, references link, and brief description of resources on human protein–protein interactions (PPIs)
and protein–DNA interactions (PDIs)
Name, acronym (reference) Web link Full name and description
2P2Idb (Bourgeas, http://www.hupo.org/research/hpp/ Hand-curated database dedicated
Basse, Morelli, & Roche, 2010) to the structure of protein–protein
complexes with known small molecule
inhibitors
3did (Mosca, Ceol, http://www.3did.irbbarcelona.org/ 3D interacting domains. Domain–
Stein, Olivella, & Aloy, 2013) domain interactions in proteins with
known 3D structures.
3D-Interologs (Lo, Chen, & Yang, http://gemdock.life.nctu.edu.tw/3d-interologs/ Protein–protein interactions in various
2010) evolutionary lineages
AANT (Hoffman et al., 2004) http://aant.icmb.utexas.edu/ Amino acid–nucleotide interaction
database. Categorizes all amino
acid–nucleotide interactions from
experimentally determined
protein–nucleic acid structures, and
provides users with a graphic interface
for visualizing these interactions in
aggregate.
BioGRID (Chatr-Aryamontri http://thebiogrid.org/ Genetic and physical interactions
et al., 2013) in yeast, worm, fly, and human
BioLiP (Yang, Roy, http://zhanglab.ccmb.med.umich.edu/BioLiP/ Semi-manually curated database for
& Zhang, 2013) high-quality, biologically relevant
ligand–protein-binding interactions
CancerResource (Ahmed et al., http://bioinf-data.charite.de/cancerresource/ Cancer-relevant proteins and
2011) compound interactions
CCSB Interactome database http://interactome.dfci.harvard.edu/index.php? Database including data from different
(reference not available to cite page¼home organisms, including humans
this database)
CellCircuits (Mak, Daly, Gruebel, http://www.cellcircuits.org/search/index.html Molecular network models: from
& Ideker, 2007) pairwise molecular interactions to
whole pathways
ConsensusPathDB (Kamburov, http://cpdb.molgen.mpg.de/ Integrates interaction networks in
Stelzl, Lehrach, & Herwig, 2013) Homo sapiens including binary and
complex protein–protein interactions,
genetic, metabolic, signaling,
gene regulatory, and
drug-target interactions, as well as
biochemical pathways
HPRD (Muthusamy, Thomas, http://www.hprd.org/ Human protein reference database.
Prasad, & Pandey, 2013) http://www.humanproteinpedia.org/ Includes abundant information on
protein characterization, mass
spectrometry, and protein–protein
interaction as follows:
Coimmunoprecipitation and mass
spectrometry-based protein–protein
interaction, coimmunoprecipitation,
and Western blotting based protein–
protein interaction, fluorescence-based
experiments, immunohistochemistry,
mass spectrometric analysis, protein,
and peptide microarray, Western
blotting, yeast two-hybrid based
protein–protein interaction.
Continued
Table 8.5 Name, references link, and brief description of resources on human protein–protein interactions (PPIs)
and protein–DNA interactions (PDIs)—cont'd
Name, acronym (reference) Web link Full name and description
IntAct (Kerrien et al., 2012) http://www.ebi.ac.uk/intact/main.xhtml Database and analysis tools for protein
interaction data. All interactions are
derived from literature curation or
direct user submissions and are freely
available.
JASPAR (Portales-Casamar et al., http://jaspar.genereg.net Open-access database for eukaryotic
2010) transcription factor-binding profiles
KDBI ( Ji et al., 2003) http://bidd.nus.edu.sg/group/kdbi/kdbi.asp Kinetic data of bio-molecular
interactions database. Kinetic data of
bio-molecular interaction (KDBI) is a
collection of experimentally
determined kinetic data of protein–
protein, protein–RNA, protein–DNA,
protein–ligand, RNA–ligand, DNA–
ligand binding, or reaction events
described in the literature.
KUPS (Chen, Jeong, http://www.ittc.ku.edu/chenlab/kups/ The University of Kansas Proteomics
& Dermyer, 2011) Service (KUPS) provides high-quality
protein–protein interaction (PPI)
datasets for researchers who are
interested in elucidating PPIs with
in silico methods.
MatrixDB (Chautard, Fatoux- http://matrixdb.ibcp.fr/ Extracellular matrix interaction
Ardore, Ballut, Thierry-Mieg, database.
& Ricard-Blum, 2011)
MiMI (Tarcea et al., 2009) http://mimi.ncibi.org/MimiWeb/main-page.jsp The Michigan molecular interactions
database provides access to the
knowledge and data merged and
integrated from numerous protein
interactions databases.
MINT (Licata et al., 2012) http://mint.bio.uniroma2.it/mint/Welcome.do Focuses on experimentally verified
protein–protein interactions mined
from the scientific literature by expert
curators. The full MINT dataset can be
freely downloaded.
Negatome (Smialowski et al., http://mips.helmholtz-muenchen.de/proj/ppi/ Proteins and domain pairs which are
2010) negatome/ unlikely engaged in direct physical
interactions. The database currently
contains experimentally supported
noninteracting protein pairs derived
from two distinct sources: by manual
curation of literature and by analyzing
protein complexes from the PDB.
NPIDB (Spirin, Titov, http://npidb.belozersky.msu.ru/ Nucleic acid–protein interaction
Karyagina, & Alexeevski, 2007) database. Includes a collection of files in
the PDB format containing structural
information on DNA–protein and
RNA–protein complexes, and a
number of online tools for analysis of
the complexes.
Continued
Table 8.5 Name, references link, and brief description of resources on human protein–protein interactions (PPIs)
and protein–DNA interactions (PDIs)—cont'd
Name, acronym (reference) Web link Full name and description
Pathguide (Cary, Bader, & Sander, http://pathguide.org/ Provides an overview of more than 190
2005) web-accessible biological pathway and
network databases. These include
databases on metabolic pathways,
signaling pathways, transcription factor
targets, gene regulatory networks,
genetic interactions, protein–
compound interactions, and
protein–protein interactions.
PepCyber: PPep http://pepcyber.umn.edu/PPEP/ Human protein interactions mediated
(Gong et al., 2008) by phosphoprotein-binding domains.
PID (Schaefer et al., 2009) http://pid.nci.nih.gov/ Pathway interaction database.
Interactions and cellular processes
assembled into human signaling
pathways.
PINA (Cowley et al., 2012) http://cbg.garvan.unsw.edu.au/pina/ Protein interaction network analysis.
Integrated platform for protein
interaction network construction,
filtering, analysis, visualization, and
management. It integrates protein–
protein interaction data from several
public databases and builds a complete,
nonredundant protein interaction
datasets.
PIN database (Luc & Tempst, http://pin.mskcc.org/ Proteins interacting in the nucleus.
2004) Proteins interacting in nucleus
including information from human
(and yeast).
PINT http://www.bioinfodatabase.com/pint/ The protein–protein interactions
Protein–Protein Interactions thermodynamic database is a collection
Thermodynamic Database of experimental data of several
(Kumar & Gromiha, 2006) thermodynamic parameters along with
literature, sequence and structural
information, and experimental
conditions.
PIPs (McDowall, Scott, & Barton, http://www.compbio.dundee.ac.uk/www-pips/ Database of predicted human
2009) protein–protein interactions
piSite (Higurashi, Ishida, http://pisite.hgc.jp/ Database of protein interaction sites
& Kinoshita, 2009) using multiple binding states in PDB
ProtChemSI (Kalinina, http://pcidb.russelllab.org/ Protein–chemical structural
Wichmann, Apic, & Russell, 2011) interactions database includes all
existing 3D structures of complexes of
proteins with low molecular weight
ligands.
POINT (Huang et al., 2004) http://point.bioinformatics.tw/Welcome.do; Prediction of human protein–protein
jsessionid¼82EDC3103003E47353C764517501AAFE interactome
PrePPI (Zhang, Petrey, Garzon, http://genomequebec.mcgill.ca/PReMod/ Predicted transcriptional regulatory
Deng, & Honig, 2013) modules in the human genome
Continued
Table 8.5 Name, references link, and brief description of resources on human protein–protein interactions (PPIs)
and protein–DNA interactions (PDIs)—cont'd
Name, acronym (reference) Web link Full name and description
PROMISCUOUS (von Eichborn http://bioinformatics.charite.de/promiscuous/ Protein–protein and drug–protein
et al., 2011) interactions resource
PSIbase (Gong et al., 2005) http://psibase.kobic.re.kr/ Molecular interaction database focusing
on structural interaction
of proteins and their domains
SNAPPI ( Jefferson, Walsh, http://www.compbio.dundee.ac.uk/SNAPPI/ Structures, interfaces, and alignments
Roberts, & Barton, 2007) downloads.jsp for protein–protein interactions
TissueNet (Barshir et al., 2013) http://netbio.bgu.ac.il/tissuenet/ Tissue distribution of protein–protein
interactions
TRANSFAC (Wingender, 2008) http://www.biobase-international.com/product/ Private database for eukaryotic
transcription-factor-binding-sites transcription factor-binding profiles
TRIP (Shin et al., 2012) http://www.trpchannel.org/ Protein–protein interactions for
mammalian TRP channels
UniHI (Chaurasia & Futschik, http://www.unihi.org/ Integrated tool for the exploration
2012) of the human interactome
UniPROBE (Robasky & Bulyk, http://the_brain.bwh.harvard.edu/uniprobe/ Online database of protein-binding
2011) microarray data on protein–DNA
interactions
Screening of Protein–Protein and Protein–DNA Interactions 269
(Tyagi et al., 2012). For that reason, despite the increasing usability of
databases, literature searches on peer-reviewed journals still constitutes
the main source of information on PPIs in two ways: first, literature
searches may retrieve information that may not be included in databases.
Second, the comparison of the list of protein interactors retrieved upon
database and literature searches enable manual correction of involuntary
mistakes due to, for example, gene acronym redundancies (Casado-
Vela, Matthiesen, Selles, & Naranjo, 2013).
ACKNOWLEDGMENTS
J. C.-V. is a JAE-DOC (CSIC) holder supported by Ministerio de Economı́a y Competitividad,
Spain, co-funded by the European Social Fund.
Screening of Protein–Protein and Protein–DNA Interactions 271
REFERENCES
Ahmed, J., Meinel, T., Dunkel, M., Murgueitio, M. S., Adams, R., Blasse, C., et al. (2011).
CancerResource: A comprehensive database of cancer-relevant proteins and compound
interactions supported by experimental knowledge. Nucleic Acids Research, 39,
D960–D967. http://dx.doi.org/10.1093/nar/gkq910.
Anderson, K. S., & LaBaer, J. (2005). The sentinel within: Exploiting the immune system for
cancer biomarkers. Journal of Proteome Research, 4(4), 1123–1133. http://dx.doi.org/
10.1021/pr0500814.
Anderson, K. S., Ramachandran, N., Wong, J., Raphael, J. V., Hainsworth, E.,
Demirkan, G., et al. (2008). Application of protein microarrays for multiplexed detection
of antibodies to tumor antigens in breast cancer. Journal of Proteome Research, 7(4),
1490–1499. http://dx.doi.org/10.1021/pr700804c.
Anderson, K. S., Sibani, S., Wallstrom, G., Qiu, J., Mendoza, E. A., Raphael, J., et al. (2011).
Protein microarray signature of autoantibody biomarkers for the early detection of breast
cancer. Journal of Proteome Research, 10(1), 85–96. http://dx.doi.org/10.1021/pr100686b.
Angenendt, P., Kreutzberger, J., Glokler, J., & Hoheisel, J. D. (2006). Generation of high
density protein microarrays by cell-free in situ expression of unpurified PCR products.
Molecular & Cellular Proteomics, 5(9), 1658–1666. http://dx.doi.org/10.1074/mcp.
T600024-MCP200.
Aranda, B., Blankenburg, H., Kerrien, S., Brinkman, F. S., Ceol, A., Chautard, E., et al.
(2011). PSICQUIC and PSISCORE: Accessing and scoring molecular interactions.
Nature Methods, 8(7), 528–529. http://dx.doi.org/10.1038/nmeth.1637.
Arduengo, M., Schenborn, E., & Hurst, R. (2007). The role of cell-free rabbit reticulocyte
expression systems in functional proteomics. In W. A. Kudlicki, F. Katzen, &
R. P. Bennett (Eds.), Cell-free protein expression (pp. 1–18). Carlsbad, CA: Landes
Bioscience.
Aronson, J. K. (2005). Biomarkers and surrogate endpoints. British Journal of Clinical Pharma-
cology, 59(5), 491–494. http://dx.doi.org/10.1111/j.1365-2125.2005.02435.x.
Babu, M. M., Luscombe, N. M., Aravind, L., Gerstein, M., & Teichmann, S. A. (2004).
Structure and evolution of transcriptional regulatory networks. Current Opinion in Struc-
tural Biology, 14(3), 283–291. http://dx.doi.org/10.1016/j.sbi.2004.05.004.
Badis, G., Berger, M. F., Philippakis, A. A., Talukder, S., Gehrke, A. R., Jaeger, S. A., et al.
(2009). Diversity and complexity in DNA recognition by transcription factors. Science,
324(5935), 1720–1723. http://dx.doi.org/10.1126/science.1162327.
Barshir, R., Basha, O., Eluk, A., Smoly, I. Y., Lan, A., & Yeger-Lotem, E. (2013). The
TissueNet database of human tissue protein-protein interactions. Nucleic Acids Research,
41, D841–D844. http://dx.doi.org/10.1093/nar/gks1198.
Beare, P. A., Chen, C., Bouman, T., Pablo, J., Unal, B., Cockrell, D. C., et al. (2008). Can-
didate antigens for Q fever serodiagnosis revealed by immunoscreening of a Coxiella
burnetii protein microarray. Clinical and Vaccine Immunology, 15(12), 1771–1779.
http://dx.doi.org/10.1128/CVI.00300-08.
Belov, L., de la Vega, O., dos Remedios, C. G., Mulligan, S. P., & Christopherson, R. I.
(2001). Immunophenotyping of leukemias using a cluster of differentiation antibody
microarray. Cancer Research, 61(11), 4483–4489.
Belov, L., Huang, P., Barber, N., Mulligan, S. P., & Christopherson, R. I. (2003). Identi-
fication of repertoires of surface antigens on leukemias using an antibody microarray.
Proteomics, 3(11), 2147–2154. http://dx.doi.org/10.1002/pmic.200300599.
Berger, M. F., Badis, G., Gehrke, A. R., Talukder, S., Philippakis, A. A., Pena-Castillo, L.,
et al. (2008). Variation in homeodomain DNA binding revealed by high-resolution anal-
ysis of sequence preferences. Cell, 133(7), 1266–1276. http://dx.doi.org/10.1016/
j.cell.2008.05.024.
272 Juan Casado-Vela et al.
Berger, M. F., & Bulyk, M. L. (2009). Universal protein-binding microarrays for the com-
prehensive characterization of the DNA-binding specificities of transcription factors.
Nature Protocols, 4(3), 393–411. http://dx.doi.org/10.1038/nprot.2008.195.
Berger, M. F., Philippakis, A. A., Qureshi, A. M., He, F. S., Estep, P. W., 3rd., &
Bulyk, M. L. (2006). Compact, universal DNA microarrays to comprehensively deter-
mine transcription-factor binding site specificities. Nature Biotechnology, 24(11),
1429–1435. http://dx.doi.org/10.1038/nbt1246.
Blankenburg, H., Finn, R. D., Prlic, A., Jenkinson, A. M., Ramirez, F., Emig, D., et al.
(2009). DASMI: Exchanging, annotating and assessing molecular interaction data.
Bioinformatics, 25(10), 1321–1328. http://dx.doi.org/10.1093/bioinformatics/btp142.
Bourgeas, R., Basse, M. J., Morelli, X., & Roche, P. (2010). Atomic analysis of protein-
protein interfaces with known inhibitors: The 2P2I database. PLoS One, 5(3), e9598.
http://dx.doi.org/10.1371/journal.pone.0009598.
Bowen, B., Steinberg, J., Laemmli, U. K., & Weintraub, H. (1980). The detection of DNA-
binding proteins by protein blotting. Nucleic Acids Research, 8(1), 1–20. http://dx.doi.
org/10.1093/nar/8.1.1.
Brase, J. C., Mannsperger, H., Frohlich, H., Gade, S., Schmidt, C., Wiemann, S., et al.
(2010). Increasing the sensitivity of reverse phase protein arrays by antibody-mediated
signal amplification. Proteome Science, 8, 36. http://dx.doi.org/10.1186/1477-5956-8-36.
Braun, P., Tasan, M., Dreze, M., Barrios-Rodiles, M., Lemmens, I., Yu, H., et al. (2009). An
experimentally derived confidence score for binary protein-protein interactions. Nature
Methods, 6(1), 91–97. http://dx.doi.org/10.1038/nmeth.1281.
Bruckner, A., Polge, C., Lentze, N., Auerbach, D., & Schlattner, U. (2009). Yeast two-
hybrid, a powerful tool for systems biology. International Journal of Molecular Sciences,
10(6), 2763–2788. http://dx.doi.org/10.3390/ijms10062763.
Cary, M. P., Bader, G. D., & Sander, C. (2005). Pathway information for systems biology.
FEBS Letters, 579(8), 1815–1820. http://dx.doi.org/10.1016/j.febslet.2005.02.005.
Casado-Vela, J., Cebrian, A., del Pulgar, M. T., Sanchez-Lopez, E., Vilaseca, M.,
Menchen, L., et al. (2011). Lights and shadows of proteomic technologies for the study
of protein species including isoforms, splicing variants and protein post-translational
modifications. Proteomics, 11(4), 590–603. http://dx.doi.org/10.1002/pmic.201000287.
Casado-Vela, J., Cebrian, A., Gomez del Pulgar, M. T., & Lacal, J. C. (2011). Approaches for
the study of cancer: Towards the integration of genomics, proteomics and metabolomics.
Clinical & Translational Oncology, 13(9), 617–628. http://dx.doi.org/10.1007/s12094-
011-0707-9.
Casado-Vela, J., Gonzalez-Gonzalez, M., Matarraz, S., Martinez-Esteso, M., Vilella, M.,
Sayagues, J., et al. (2013). Protein arrays: Recent achievements and their application
to study the human proteome. Current Proteomics, 10(2), 83–87. http://dx.doi.org/
10.2174/1570164611310020003.
Casado-Vela, J., Lacal, J. C., & Elortza, F. (2013). Protein chimerism: Novel source of pro-
tein diversity in humans adds complexity to bottom-up proteomics. Proteomics, 13(1),
5–11. http://dx.doi.org/10.1002/pmic.201200371.
Casado-Vela, J., Martinez-Torrecuadrada, J. L., & Casal, J. I. (2009). Differential phosphor-
ylation patterns between the Cyclin-A2/CDK2 complex and their monomers. Protein
Expression and Purification, 66(1), 15–21. http://dx.doi.org/10.1016/j.pep.2009.02.007.
Casado-Vela, J., Matthiesen, R., Selles, S., & Naranjo, J. R. (2013). Protein-protein inter-
actions: Gene acronym redundancies and current limitations precluding automated data
integration. Proteomes, 1(1), 3–24. http://dx.doi.org/10.3390/proteomes1010003.
Casado-Vela, J., Rodriguez-Suarez, E., Iloro, I., Ametzazurra, A., Alkorta, N., Garcia-
Velasco, J. A., et al. (2009). Comprehensive proteomic analysis of human endometrial
fluid aspirate. Journal of Proteome Research, 8(10), 4622–4632. http://dx.doi.org/
10.1021/pr9004426.
Screening of Protein–Protein and Protein–DNA Interactions 273
Chandra, H., Reddy, P. J., & Srivastava, S. (2011). Protein microarrays and novel
detection platforms. Expert Review of Proteomics, 8(1), 61–79. http://dx.doi.org/
10.1586/epr.10.99.
Chatr-Aryamontri, A., Breitkreutz, B. J., Heinicke, S., Boucher, L., Winter, A., Stark, C.,
et al. (2013). The BioGRID interaction database: 2013 update. Nucleic Acids Research, 41,
D816–D823. http://dx.doi.org/10.1093/nar/gks1158.
Chaurasia, G., & Futschik, M. (2012). The integration and annotation of the human inter-
actome in the UniHI Database. Methods in Molecular Biology, 812, 175–188. http://dx.
doi.org/10.1007/978-1-61779-455-1_10.
Chautard, E., Fatoux-Ardore, M., Ballut, L., Thierry-Mieg, N., & Ricard-Blum, S. (2011).
MatrixDB, the extracellular matrix interaction database. Nucleic Acids Research, 39,
D235–D240. http://dx.doi.org/10.1093/nar/gkq830.
Chen, Q., Chen, J., Sun, T., Shen, J., Shen, X., & Jiang, H. (2004). A yeast two-hybrid
technology-based system for the discovery of PPARgamma agonist and antagonist. Ana-
lytical Biochemistry, 335(2), 253–259. http://dx.doi.org/10.1016/j.ab.2004.09.004.
Chen, X. W., Jeong, J. C., & Dermyer, P. (2011). KUPS: Constructing datasets of interacting
and non-interacting protein pairs with associated attributions. Nucleic Acids Research, 39,
D750–D754. http://dx.doi.org/10.1093/nar/gkq943.
Chen, Y. C., Rajagopala, S. V., Stellberger, T., & Uetz, P. (2010). Exhaustive benchmarking
of the yeast two-hybrid system. Nature Methods, 7(9), 667–668. http://dx.doi.org/
10.1038/nmeth0910-667.
Chen, J., Zhou, J., Sanders, C. K., Nolan, J. P., & Cai, H. (2009). A surface display yeast two-
hybrid screening system for high-throughput protein interactome mapping. Analytical
Biochemistry, 390(1), 29–37. http://dx.doi.org/10.1016/j.ab.2009.03.013.
Corthals, G. L., Wasinger, V. C., Hochstrasser, D. F., & Sanchez, J. C. (2000). The dynamic
range of protein expression: A challenge for proteomic research. Electrophoresis, 21(6),
1104–1115. http://dx.doi.org/10.1002/(SICI)1522-2683(20000401)21:6<1104::AID-
ELPS1104>3.0.CO;2-C.
Cowley, M. J., Pinese, M., Kassahn, K. S., Waddell, N., Pearson, J. V., Grimmond, S. M.,
et al. (2012). PINA v2.0: Mining interactome modules. Nucleic Acids Research, 40,
D862–D865. http://dx.doi.org/10.1093/nar/gkr967.
Dı́ez, P., Dasilva, N., Gonzalez-Gonzalez, M., Matarraz, S., Casado-Vela, J., Orfao, A., et al.
(2012). Data analysis strategies for protein microarrays. Microarrays, 1, 64–83. http://dx.
doi.org/10.3390/microarrays1020064.
Ekins, R., & Chu, F. (1992). Multianalyte microspot immunoassay. The microanalytical
‘compact disk’ of the future. Annales de Biologie Clinique, 50(5), 337–353.
Ekins, R., Chu, F., & Biggart, E. (1990). Multispot, multianalyte, immunoassay. Annales de
Biologie Clinique, 48(9), 655–666.
Ellmark, P., Belov, L., Huang, P., Lee, C. S., Solomon, M. J., Morgan, D. K., et al. (2006).
Multiplex detection of surface molecules on colorectal cancers. Proteomics, 6(6),
1791–1802. http://dx.doi.org/10.1002/pmic.200500468.
Espina, V., Mehta, A. I., Winters, M. E., Calvert, V., Wulfkuhle, J., Petricoin, E. F., et al.
(2003). Protein microarrays: Molecular profiling technologies for clinical specimens.
Proteomics, 3(11), 2091–2100. http://dx.doi.org/10.1002/pmic.200300592.
Feijs, K. L., Kleine, H., Braczynski, A., Forst, A. H., Herzog, N., Verheugd, P., et al. (2013).
ARTD10 substrate identification on protein microarrays: Regulation of GSK3beta by
mono-ADP-ribosylation. Cell Communication and Signaling, 11(1), 5. http://dx.doi.
org/10.1186/1478-811X-11-5.
Feilner, T., Hultschig, C., Lee, J., Meyer, S., Immink, R. G., Koenig, A., et al. (2005). High
throughput identification of potential Arabidopsis mitogen-activated protein kinases
substrates. Molecular & Cellular Proteomics, 4(10), 1558–1568. http://dx.doi.org/
10.1074/mcp.M500007-MCP200.
274 Juan Casado-Vela et al.
Feldmann, M. (2008). Many cytokines are very useful therapeutic targets in disease. The Jour-
nal of Clinical Investigation, 118(11), 3533–3536. http://dx.doi.org/10.1172/JCI37346.
Fenner, B. J., Scannell, M., & Prehn, J. H. (2010). Expanding the substantial interactome of
NEMO using protein microarrays. PLoS One, 5(1), e8799. http://dx.doi.org/10.1371/
journal.pone.0008799.
Fernandez-Suarez, X. M., & Galperin, M. Y. (2013). The 2013 Nucleic Acids Research
Database Issue and the online molecular biology database collection. Nucleic Acids
Research, 41, D1–D7. http://dx.doi.org/10.1093/nar/gks1297.
Fields, S., & Song, O. (1989). A novel genetic system to detect protein-protein interactions.
Nature, 340(6230), 245–246. http://dx.doi.org/10.1038/340245a0.
Franco-Zorrilla, J. M., & Solano, R. (2014). High-throughput analysis of protein-DNA
binding affinity. Methods in Molecular Biology, 1062, 697–709. http://dx.doi.org/
10.1007/978-1-62703-580-4_36.
Frenkel-Morgenstern, M., Lacroix, V., Ezkurdia, I., Levin, Y., Gabashvili, A., Prilusky, J.,
et al. (2012). Chimeras taking shape: Potential functions of proteins encoded by chimeric
RNA transcripts. Genome Research, 22(7), 1231–1242. http://dx.doi.org/10.1101/
gr.130062.111.
Furney, S. J., Higgins, D. G., Ouzounis, C. A., & Lopez-Bigas, N. (2006). Structural and
functional properties of genes involved in human cancer. BMC Genomics, 7, 3. http://
dx.doi.org/10.1186/1471-2164-7-3.
Garcia-Garcia, J., Schleker, S., Klein-Seetharaman, J., & Oliva, B. (2012). BIPS: BIANA
Interolog Prediction Server. A tool for protein-protein interaction inference. Nucleic
Acids Research, 40, W147–W151. http://dx.doi.org/10.1093/nar/gks553.
Godoy, M., Franco-Zorrilla, J. M., Perez-Perez, J., Oliveros, J. C., Lorenzo, O., &
Solano, R. (2011). Improved protein-binding microarrays for the identification of
DNA-binding specificities of transcription factors. The Plant Journal, 66(4), 700–711.
http://dx.doi.org/10.1111/j.1365-313X.2011.04519.x.
Gomez, S. M., Choi, K., & Wu, Y. (2008). Prediction of protein-protein interaction
networks. Current Protocols in Bioinformatics. 8, 8.2.1–8.2.14. http://dx.doi.org/
10.1002/0471250953.bi0802s22, Chapter 8, Unit 8.2.
Gong, S., Yoon, G., Jang, I., Bolser, D., Dafas, P., Schroeder, M., et al. (2005). PSIbase:
A database of Protein Structural Interactome map (PSIMAP). Bioinformatics, 21(10),
2541–2543. http://dx.doi.org/10.1093/bioinformatics/bti366.
Gong, W., Zhou, D., Ren, Y., Wang, Y., Zuo, Z., Shen, Y., et al. (2008). PepCyber:P PEP:
A database of human protein protein interactions mediated by phosphoprotein-binding
domains. Nucleic Acids Research, 36, D679–D683. http://dx.doi.org/10.1093/nar/
gkm854.
Grove, C. A., De Masi, F., Barrasa, M. I., Newburger, D. E., Alkema, M. J., Bulyk, M. L.,
et al. (2009). A multiparameter network reveals extensive divergence between C. elegans
bHLH transcription factors. Cell, 138(2), 314–327. http://dx.doi.org/10.1016/
j.cell.2009.04.058.
Gujral, T. S., Karp, R. L., Finski, A., Chan, M., Schwartz, P. E., Macbeath, G., et al. (2012).
Profiling phospho-signaling networks in breast cancer using reverse-phase protein arrays.
Oncogene, 32, 3470–3476. http://dx.doi.org/10.1038/onc.2012.378.
Gulmann, C., Sheehan, K. M., Kay, E. W., Liotta, L. A., & Petricoin, E. F. (2006).
Array-based proteomics: Mapping of protein circuitries for diagnostics, prognostics,
and therapy guidance in cancer. The Journal of Pathology, 208(5), 595–606. http://dx.
doi.org/10.1002/path.1958.
Haab, B. B., Dunham, M. J., & Brown, P. O. (2001). Protein microarrays for highly parallel
detection and quantitation of specific proteins and antibodies in complex solutions.
Genome Biology, 2(2), 1–13.
Screening of Protein–Protein and Protein–DNA Interactions 275
Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., & Sakaki, Y. (2001).
A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proceed-
ings of the National Academy of Sciences of the United States of America, 98(8), 4569–4574.
http://dx.doi.org/10.1073/pnas.061034498.
Jansen, C., Gronenborn, A. M., & Clore, G. M. (1987). The binding of the cyclic AMP
receptor protein to synthetic DNA sites containing permutations in the consensus
sequence TGTGA. The Biochemical Journal, 246(1), 227–232.
Jefferson, E. R., Walsh, T. P., Roberts, T. J., & Barton, G. J. (2007). SNAPPI-DB:
A database and API of Structures, interfaces and alignments for protein-protein interac-
tions. Nucleic Acids Research, 35, D580–D589. http://dx.doi.org/10.1093/nar/gkl836.
Jeong, J. S., Jiang, L., Albino, E., Marrero, J., Rho, H. S., Hu, J., et al. (2012). Rapid identification
of monospecific monoclonal antibodies using a human proteome microarray. Molecular &
Cellular Proteomics. 11(6), O111.016253. http://dx.doi.org/10.1074/mcp.O111.016253.
Jessulat, M., Pitre, S., Gui, Y., Hooshyar, M., Omidi, K., Samanfar, B., et al. (2011). Recent
advances in protein-protein interaction prediction: Experimental and computational
methods. Expert Opinion on Drug Discovery, 6(9), 921–935. http://dx.doi.org/
10.1517/17460441.2011.603722.
Ji, Z. L., Chen, X., Zhen, C. J., Yao, L. X., Han, L. Y., Yeo, W. K., et al. (2003). KDBI:
Kinetic Data of Bio-molecular interactions database. Nucleic Acids Research, 31(1),
255–257. http://dx.doi.org/10.1093/nar/gkg067.
Jolma, A., Kivioja, T., Toivonen, J., Cheng, L., Wei, G., Enge, M., et al. (2010). Multiplexed
massively parallel SELEX for characterization of human transcription factor binding spec-
ificities. Genome Research, 20(6), 861–873. http://dx.doi.org/10.1101/gr.100552.109.
Jungblut, P., Thiede, B., Zimny-Arndt, U., Muller, E. C., Scheler, C., Wittmann-
Liebold, B., et al. (1996). Resolution power of two-dimensional electrophoresis and
identification of proteins from gels. Electrophoresis, 17(5), 839–847. http://dx.doi.org/
10.1002/elps.1150170505.
Kader, H. A., Tchernev, V. T., Satyaraj, E., Lejnine, S., Kotler, G., Kingsmore, S. F., et al.
(2005). Protein microarray analysis of disease activity in pediatric inflammatory bowel
disease demonstrates elevated serum PLGF, IL-7, TGF-beta1, and IL-12p40 levels in
Crohn’s disease and ulcerative colitis patients in remission versus active disease. The
American Journal of Gastroenterology, 100(2), 414–423. http://dx.doi.org/10.1111/
j.1572-0241.2005.40819.x.
Kalinina, O. V., Wichmann, O., Apic, G., & Russell, R. B. (2011). Combinations of
protein-chemical complex structures reveal new targets for established drugs. PLoS Com-
putational Biology, 7(5), e1002043. http://dx.doi.org/10.1371/journal.pcbi.1002043.
Kamburov, A., Stelzl, U., Lehrach, H., & Herwig, R. (2013). The ConsensusPathDB inter-
action database: 2013 update. Nucleic Acids Research, 41, D793–D800. http://dx.doi.org/
10.1093/nar/gks1055.
Katz, C., Levy-Beladev, L., Rotem-Bamberger, S., Rito, T., Rudiger, S. G., & Friedler, A.
(2011). Studying protein-protein interactions using peptide arrays. Chemical Society
Reviews, 40(5), 2131–2145. http://dx.doi.org/10.1039/c0cs00029a.
Katzen, F., Chang, G., & Kudlicki, W. (2005). The past, present and future of cell-free pro-
tein synthesis. Trends in Biotechnology, 23(3), 150–156. http://dx.doi.org/10.1016/
j.tibtech.2005.01.003.
Kerrien, S., Aranda, B., Breuza, L., Bridge, A., Broackes-Carter, F., Chen, C., et al. (2012).
The IntAct molecular interaction database in 2012. Nucleic Acids Research, 40,
D841–D846. http://dx.doi.org/10.1093/nar/gkr1088.
Kersten, B., Possling, A., Blaesing, F., Mirgorodskaya, E., Gobom, J., & Seitz, H. (2004).
Protein microarray technology and ultraviolet crosslinking combined with mass spec-
trometry for the analysis of protein-DNA interactions. Analytical Biochemistry, 331(2),
303–313. http://dx.doi.org/10.1016/j.ab.2004.05.008.
Screening of Protein–Protein and Protein–DNA Interactions 277
Kim, D., Daniel, W. L., & Mirkin, C. A. (2009). Microarray-based multiplexed scanometric
immunoassay for protein cancer markers using gold nanoparticle probes. Analytical
Chemistry, 81(21), 9183–9187. http://dx.doi.org/10.1021/ac9018389.
Kim, M. J., Lee, T. H., Pahk, Y. M., Kim, Y. H., Park, H. M., Choi, Y. D., et al. (2009).
Quadruple 9-mer-based protein binding microarray with DsRed fusion protein. BMC
Molecular Biology, 10, 91. http://dx.doi.org/10.1186/1471-2199-10-91.
Klingstrom, T., & Plewczynski, D. (2011). Protein-protein interaction and pathway data-
bases, a graphical review. Briefings in Bioinformatics, 12(6), 702–713. http://dx.doi.org/
10.1093/bib/bbq064.
Kumar, M. D., & Gromiha, M. M. (2006). PINT: Protein-protein interactions thermodynamic
database. Nucleic Acids Research, 34, D195–D198. http://dx.doi.org/10.1093/nar/gkj017.
Kusnezow, W., Jacob, A., Walijew, A., Diehl, F., & Hoheisel, J. D. (2003). Antibody micro-
arrays: An evaluation of production parameters. Proteomics, 3(3), 254–264. http://dx.doi.
org/10.1002/pmic.200390038.
LaBaer, J., & Ramachandran, N. (2005). Protein microarrays as tools for functional proteo-
mics. Current Opinion in Chemical Biology, 9(1), 14–19. http://dx.doi.org/10.1016/
j.cbpa.2004.12.006.
Lane, D., Prentki, P., & Chandler, M. (1992). Use of gel retardation to analyze protein-
nucleic acid interactions. Microbiological Reviews, 56(4), 509–528.
Lange, V., Picotti, P., Domon, B., & Aebersold, R. (2008). Selected reaction monitoring for
quantitative proteomics: A tutorial. Molecular Systems Biology, 4, 222. http://dx.doi.org/
10.1038/msb.2008.61.
Lee, J. R., Magee, D. M., Gaster, R. S., LaBaer, J., & Wang, S. X. (2013). Emerging protein
array technologies for proteomics. Expert Review of Proteomics, 10(1), 65–75. http://dx.
doi.org/10.1586/epr.12.67.
Li, H., Wang, J., Mor, G., & Sklar, J. (2008). A neoplastic gene fusion mimics trans-splicing
of RNAs in normal human cells. Science, 321(5894), 1357–1361. http://dx.doi.org/
10.1126/science.1156725.
Licata, L., Briganti, L., Peluso, D., Perfetto, L., Iannuccelli, M., Galeota, E., et al. (2012).
MINT, the molecular interaction database: 2012 update. Nucleic Acids Research, 40,
D857–D861. http://dx.doi.org/10.1093/nar/gkr930.
Liotta, L. A., Espina, V., Mehta, A. I., Calvert, V., Rosenblatt, K., Geho, D., et al. (2003).
Protein microarrays: Meeting analytical challenges for clinical applications. Cancer Cell,
3(4), 317–325. http://dx.doi.org/10.1016/S1535-6108(03)00086-2.
Lo, Y. S., Chen, Y. C., & Yang, J. M. (2010). 3D-interologs: An evolution database of phys-
ical protein- protein interactions across multiple genomes. BMC Genomics, 11(Suppl 3),
S7. http://dx.doi.org/10.1186/1471-2164-11-S3-S7.
Locker, G. J., Steger, G. G., Gnant, M. F., Steiner, B., Simonitsch, I., Krainer, M., et al.
(1999). Induction of immunomediated diseases by recombinant human granulocyte-
macrophage colony-stimulating factor during cancer treatment? Journal of Immunotherapy,
22(1), 85–89.
Luc, P. V., & Tempst, P. (2004). PINdb: A database of nuclear protein complexes from
human and yeast. Bioinformatics, 20(9), 1413–1415. http://dx.doi.org/10.1093/bioinfor-
matics/bth114.
Ly, L., & Wasinger, V. C. (2011). Protein and peptide fractionation, enrichment and deple-
tion: Tools for the complex proteome. Proteomics, 11(4), 513–534. http://dx.doi.org/
10.1002/pmic.201000394.
MacBeath, G., & Schreiber, S. L. (2000). Printing proteins as microarrays for high-
throughput function determination. Science, 289(5485), 1760–1763. http://dx.doi.
org/10.1126/science.289.5485.1760.
Madoz-Gurpide, J., Kuick, R., Wang, H., Misek, D. E., & Hanash, S. M. (2008). Integral
protein microarrays for the identification of lung cancer antigens in sera that induce a
278 Juan Casado-Vela et al.
humoral immune response. Molecular & Cellular Proteomics, 7(2), 268–281. http://dx.doi.
org/10.1074/mcp.M700366-MCP200.
Mak, H. C., Daly, M., Gruebel, B., & Ideker, T. (2007). Cell Circuits: A database of protein
network models. Nucleic Acids Research, 35, D538–D545. http://dx.doi.org/10.1093/
nar/gkl937.
Matarraz, S., Gonzalez-Gonzalez, M., Jara, M., Orfao, A., & Fuentes, M. (2011). New tech-
nologies in cancer. Protein microarrays for biomarker discovery. Clinical & Translational
Oncology, 13(3), 156–161. http://dx.doi.org/10.1007/s12094-011-0635-8.
McDowall, M. D., Scott, M. S., & Barton, G. J. (2009). PIPs: Human protein-protein inter-
action prediction database. Nucleic Acids Research, 37, D651–D656. http://dx.doi.org/
10.1093/nar/gkn870.
McInnes, I. B., & Schett, G. (2007). Cytokines in the pathogenesis of rheumatoid arthritis.
Nature Reviews. Immunology, 7(6), 429–442. http://dx.doi.org/10.1038/nri2094.
McKenna, N. J., & O’Malley, B. W. (2002). Combinatorial control of gene expression by
nuclear receptors and coregulators. Cell, 108(4), 465–474.
Meng, X., Brodsky, M. H., & Wolfe, S. A. (2005). A bacterial one-hybrid system for deter-
mining the DNA-binding specificity of transcription factors. Nature Biotechnology, 23(8),
988–994. http://dx.doi.org/10.1038/nbt1120.
Mijakovic, I., & Macek, B. (2012). Impact of phosphoproteomics on studies of bacterial
physiology. FEMS Microbiology Reviews, 36(4), 877–892. http://dx.doi.org/10.1111/
j.1574-6976.2011.00314.x.
Miller, J. C., Butler, E. B., Teh, B. S., & Haab, B. B. (2001). The application of protein micro-
arrays to serum diagnostics: Prostate cancer as a test case. Disease Markers, 17(4), 225–234.
Millioni, R., Tolin, S., Puricelli, L., Sbrignadello, S., Fadini, G. P., Tessari, P., et al. (2011).
High abundance proteins depletion vs low abundance proteins enrichment: Comparison
of methods to reduce the plasma proteome complexity. PLoS One, 6(5), e19603. http://
dx.doi.org/10.1371/journal.pone.0019603.
Mishra, S. (2012). Computational prediction of protein-protein complexes. BMC Research
Notes, 5(1), 495. http://dx.doi.org/10.1186/1756-0500-5-495.
Miskimins, W. K., Roberts, M. P., McClelland, A., & Ruddle, F. H. (1985). Use of a
protein-blotting procedure and a specific DNA probe to identify nuclear proteins that
recognize the promoter region of the transferrin receptor gene. Proceedings of the National
Academy of Sciences of the United States of America, 82(20), 6741–6744.
Mor, G., Visintin, I., Lai, Y., Zhao, H., Schwartz, P., Rutherford, T., et al. (2005). Serum
protein markers for early detection of ovarian cancer. Proceedings of the National Academy of
Sciences of the United States of America, 102(21), 7677–7682. http://dx.doi.org/10.1073/
pnas.0502178102.
Mosca, R., Ceol, A., Stein, A., Olivella, R., & Aloy, P. (2013). 3did: A catalog of domain-
based interactions of known three-dimensional structure. Nucleic Acids Research, 42,
D374–D379. http://dx.doi.org/10.1093/nar/gkt887.
Mukherjee, S., Berger, M. F., Jona, G., Wang, X. S., Muzzey, D., Snyder, M., et al. (2004).
Rapid analysis of the DNA-binding specificities of transcription factors with DNA
microarrays. Nature Genetics, 36(12), 1331–1339. http://dx.doi.org/10.1038/ng1473.
Muthusamy, B., Thomas, J. K., Prasad, T. S., & Pandey, A. (2013). Access guide to human
proteinpedia. Current Protocols in Bioinformatics, 41, 1–15. http://dx.doi.org/
10.1002/0471250953.bi0121s41, Chapter 1, Unit 1 21.
Nielsen, U. B., Cardone, M. H., Sinskey, A. J., MacBeath, G., & Sorger, P. K. (2003).
Profiling receptor tyrosine kinase activation by using Ab microarrays. Proceedings of the
National Academy of Sciences of the United States of America, 100(16), 9330–9335. http://
dx.doi.org/10.1073/pnas.1633513100.
Park, P. J. (2009). ChIP-seq: Advantages and challenges of a maturing technology. Nature
Reviews. Genetics, 10(10), 669–680. http://dx.doi.org/10.1038/nrg2641.
Screening of Protein–Protein and Protein–DNA Interactions 279
Paweletz, C. P., Charboneau, L., Bichsel, V. E., Simone, N. L., Chen, T., Gillespie, J. W.,
et al. (2001). Reverse phase protein microarrays which capture disease progression show
activation of pro-survival pathways at the cancer invasion front. Oncogene, 20(16),
1981–1989. http://dx.doi.org/10.1038/sj.onc.1204265.
Picotti, P., Bodenmiller, B., Mueller, L. N., Domon, B., & Aebersold, R. (2009). Full
dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell, 138(4),
795–806. http://dx.doi.org/10.1016/j.cell.2009.05.051.
Pitre, S., Alamgir, M., Green, J. R., Dumontier, M., Dehne, F., & Golshani, A. (2008).
Computational methods for predicting protein-protein interactions. Advances in Biochem-
ical Engineering/Biotechnology, 110, 247–267. http://dx.doi.org/10.1007/10_2007_089.
Portales-Casamar, E., Thongjuea, S., Kwon, A. T., Arenillas, D., Zhao, X., Valen, E., et al.
(2010). JASPAR 2010: The greatly expanded open-access database of transcription factor
binding profiles. Nucleic Acids Research, 38, D105–D110. http://dx.doi.org/10.1093/
nar/gkp950.
Ptacek, J., Devgan, G., Michaud, G., Zhu, H., Zhu, X., Fasolo, J., et al. (2005). Global anal-
ysis of protein phosphorylation in yeast. Nature, 438(7068), 679–684. http://dx.doi.org/
10.1038/nature04187.
Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, S., Rosen, B., Lau, A. Y., et al.
(2004). Self-assembling protein microarrays. Science, 305(5680), 86–90. http://dx.doi.
org/10.1126/science.1097639.
Ramachandran, N., Raphael, J. V., Hainsworth, E., Demirkan, G., Fuentes, M. G.,
Rolfs, A., et al. (2008). Next-generation high-density self-assembling functional protein
arrays. Nature Methods, 5(6), 535–538. http://dx.doi.org/10.1038/nmeth.1210.
Ramani, A. K., Bunescu, R. C., Mooney, R. J., & Marcotte, E. M. (2005). Consolidating the
set of known human protein-protein interactions in preparation for large-scale mapping
of the human interactome. Genome Biology, 6(5), R40. http://dx.doi.org/10.1186/
gb-2005-6-5-r40.
Ravasi, T., Suzuki, H., Cannistraci, C. V., Katayama, S., Bajic, V. B., Tan, K., et al. (2010).
An atlas of combinatorial transcriptional regulation in mouse and man. Cell, 140(5),
744–752. http://dx.doi.org/10.1016/j.cell.2010.01.044.
Remenyi, A., Scholer, H. R., & Wilmanns, M. (2004). Combinatorial control of gene
expression. Nature Structural & Molecular Biology, 11(9), 812–815. http://dx.doi.org/
10.1038/nsmb820.
Robasky, K., & Bulyk, M. L. (2011). UniPROBE, update 2011: Expanded content and search
tools in the online database of protein-binding microarray data on protein-DNA interac-
tions. Nucleic Acids Research, 39, D124–D128. http://dx.doi.org/10.1093/nar/gkq992.
Roulet, E., Busso, S., Camargo, A. A., Simpson, A. J., Mermod, N., & Bucher, P. (2002).
High-throughput SELEX SAGE method for quantitative modeling of transcription-factor
binding sites. Nature Biotechnology, 20(8), 831–835. http://dx.doi.org/10.1038/nbt718.
Sahdev, S., Khattar, S. K., & Saini, K. S. (2008). Production of active eukaryotic proteins
through bacterial expression systems: A review of the existing biotechnology strategies.
Molecular and Cellular Biochemistry, 307(1–2), 249–264. http://dx.doi.org/10.1007/
s11010-007-9603-6.
Schaefer, C. F., Anthony, K., Krupa, S., Buchoff, J., Day, M., Hannay, T., et al. (2009). PID:
The Pathway Interaction Database. Nucleic Acids Research, 37, D674–D679. http://dx.
doi.org/10.1093/nar/gkn653.
Schleker, S., Sun, J., Raghavan, B., Srnec, M., Muller, N., Koepfinger, M., et al. (2012). The
current Salmonella-host interactome. Proteomics. Clinical Applications, 6(1–2), 117–133.
http://dx.doi.org/10.1002/prca.201100083.
Schwarz, D., Dotsch, V., & Bernhard, F. (2008). Production of membrane proteins using
cell-free expression systems. Proteomics, 8(19), 3933–3946. http://dx.doi.org/10.1002/
pmic.200800171.
280 Juan Casado-Vela et al.
Schweitzer, B., Predki, P., & Snyder, M. (2003). Microarrays to characterize protein inter-
actions on a whole-proteome scale. Proteomics, 3(11), 2190–2199. http://dx.doi.org/
10.1002/pmic.200300610.
Shin, Y. C., Shin, S. Y., Chun, J. N., Cho, H. S., Lim, J. M., Kim, H. G., et al. (2012). TRIP
database 2.0: A manually curated information hub for accessing TRP channel interaction
network. PLoS One, 7(10), e47165. http://dx.doi.org/10.1371/journal.pone.0047165.
Sibani, S., & LaBaer, J. (2011). Immunoprofiling using NAPPA protein microarrays. Methods
in Molecular Biology, 723, 149–161. http://dx.doi.org/10.1007/978-1-61779-043-0_10.
Skrabanek, L., Saini, H. K., Bader, G. D., & Enright, A. J. (2008). Computational prediction
of protein-protein interactions. Molecular Biotechnology, 38(1), 1–17. http://dx.doi.org/
10.1007/s12033-007-0069-2.
Smialowski, P., Pagel, P., Wong, P., Brauner, B., Dunger, I., Fobo, G., et al. (2010). The
Negatome database: A reference set of non-interacting protein pairs. Nucleic Acids
Research, 38, D540–D544. http://dx.doi.org/10.1093/nar/gkp1026.
Spirin, S., Titov, M., Karyagina, A., & Alexeevski, A. (2007). NPIDB: A database of nucleic
acids-protein interactions. Bioinformatics, 23(23), 3247–3248. http://dx.doi.org/
10.1093/bioinformatics/btm519.
Spurrier, B., Ramalingam, S., & Nishizuka, S. (2008). Reverse-phase protein lysate micro-
arrays for cell signaling analysis. Nature Protocols, 3(11), 1796–1808. http://dx.doi.org/
10.1038/nprot.2008.179.
Stumpf, M. P., Thorne, T., de Silva, E., Stewart, R., An, H. J., Lappe, M., et al. (2008).
Estimating the size of the human interactome. Proceedings of the National Academy of Sci-
ences of the United States of America, 105(19), 6959–6964. http://dx.doi.org/10.1073/
pnas.0708078105.
Tao, S. C., & Zhu, H. (2006). Protein chip fabrication by capture of nascent polypeptides.
Nature Biotechnology, 24(10), 1253–1254. http://dx.doi.org/10.1038/nbt1249.
Tarcea, V. G., Weymouth, T., Ade, A., Bookvich, A., Gao, J., Mahavisno, V., et al. (2009).
Michigan molecular interactions r2: From interacting proteins to pathways. Nucleic Acids
Research, 37, D642–D646. http://dx.doi.org/10.1093/nar/gkn722.
Tarrant, M. K., & Cole, P. A. (2009). The chemical biology of protein phosphorylation.
Annual Review of Biochemistry, 78, 797–825. http://dx.doi.org/10.1146/annurev.
biochem.78.070907.103047.
Thaxton, C. S., Elghanian, R., Thomas, A. D., Stoeva, S. I., Lee, J. S., Smith, N. D., et al.
(2009). Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and bio-
chemical recurrence after radical prostatectomy. Proceedings of the National Academy of Sci-
ences of the United States of America, 106(44), 18437–18442. http://dx.doi.org/10.1073/
pnas.0904719106.
Tran, N. H., Choi, K. P., & Zhang, L. (2013). Counting motifs in the human interactome.
Nature Communications, 4, 2241. http://dx.doi.org/10.1038/ncomms3241.
Tyagi, M., Hashimoto, K., Shoemaker, B. A., Wuchty, S., & Panchenko, A. R. (2012).
Large-scale mapping of human protein interactome using structural complexes. EMBO
Reports, 13(3), 266–271. http://dx.doi.org/10.1038/embor.2011.261.
Vaquerizas, J. M., Kummerfeld, S. K., Teichmann, S. A., & Luscombe, N. M. (2009).
A census of human transcription factors: Function, expression and evolution. Nature
Reviews. Genetics, 10(4), 252–263. http://dx.doi.org/10.1038/nrg2538.
Venkatesan, K., Rual, J. F., Vazquez, A., Stelzl, U., Lemmens, I., Hirozane-Kishikawa, T.,
et al. (2009). An empirical framework for binary interactome mapping. Nature Methods,
6(1), 83–90. http://dx.doi.org/10.1038/nmeth.1280.
Vigil, A., Chen, C., Jain, A., Nakajima-Sasaki, R., Jasinskas, A., Pablo, J., et al. (2011). Pro-
filing the humoral immune response of acute and chronic Q fever by protein microarray.
Molecular & Cellular Proteomics, 10(10), M110.006304. http://dx.doi.org/10.1074/mcp.
M110.006304.
Screening of Protein–Protein and Protein–DNA Interactions 281
Vigil, A., Ortega, R., Nakajima-Sasaki, R., Pablo, J., Molina, D. M., Chao, C. C., et al.
(2010). Genome-wide profiling of humoral immune response to Coxiella burnetii infec-
tion by protein microarray. Proteomics, 10(12), 2259–2269. http://dx.doi.org/10.1002/
pmic.201000064.
Virok, D. P., Simon, D., Bozso, Z., Rajko, R., Datki, Z., Balint, E., et al. (2011). Protein
array based interactome analysis of amyloid-beta indicates an inhibition of protein trans-
lation. Journal of Proteome Research, 10(4), 1538–1547. http://dx.doi.org/10.1021/
pr1009096.
von Eichborn, J., Murgueitio, M. S., Dunkel, M., Koerner, S., Bourne, P. E., & Preissner, R.
(2011). PROMISCUOUS: A database for network-based drug-repositioning. Nucleic
Acids Research, 39, D1060–D1066. http://dx.doi.org/10.1093/nar/gkq1037.
Wang, L., Cole, K. D., He, H. J., Hancock, D. K., Gaigalas, A. K., & Zong, Y. (2006). Com-
parison of ovalbumin quantification using forward-phase protein microarrays and sus-
pension arrays. Journal of Proteome Research, 5(7), 1770–1775. http://dx.doi.org/
10.1021/pr060074v.
Warren, C. L., Kratochvil, N. C., Hauschild, K. E., Foister, S., Brezinski, M. L.,
Dervan, P. B., et al. (2006). Defining the sequence-recognition profile of DNA-binding
molecules. Proceedings of the National Academy of Sciences of the United States of America,
103(4), 867–872. http://dx.doi.org/10.1073/pnas.0509843102.
Wilson, B., Liotta, L. A., & Petricoin, E. F. (2010). Monitoring proteins and protein net-
works using reverse phase protein arrays. Disease Markers, 28(4), 225–232. http://dx.
doi.org/10.3233/DMA-2010-0705.
Wingender, E. (2008). The TRANSFAC project as an example of framework technology
that supports the analysis of genomic regulation. Briefings in Bioinformatics, 9(4),
326–332. http://dx.doi.org/10.1093/bib/bbn016.
Wingender, E., Chen, X., Fricke, E., Geffers, R., Hehl, R., Liebich, I., et al. (2001). The
TRANSFAC system on gene expression regulation. Nucleic Acids Research, 29(1),
281–283.
Wingren, C., & Borrebaeck, C. A. (2009). Antibody-based microarrays. Methods in Molecular
Biology, 509, 57–84. http://dx.doi.org/10.1007/978-1-59745-372-1_5.
Woodbury, C. P., Jr., & von Hippel, P. H. (1983). On the determination of
deoxyribonucleic acid-protein interaction parameters using the nitrocellulose filter-
binding assay. Biochemistry, 22(20), 4730–4737. http://dx.doi.org/10.1021/
bi00289a018.
Xu, X., Song, Y., Li, Y., Chang, J., Zhang, H., & An, L. (2010). The tandem affinity puri-
fication method: An efficient system for protein complex purification and protein inter-
action identification. Protein Expression and Purification, 72(2), 149–156. http://dx.doi.
org/10.1016/j.pep.2010.04.009.
Yang, J., Roy, A., & Zhang, Y. (2013). BioLiP: A semi-manually curated database for bio-
logically relevant ligand-protein interactions. Nucleic Acids Research, 41, D1096–D1103.
http://dx.doi.org/10.1093/nar/gks966.
Yarden, Y., & Sliwkowski, M. X. (2001). Untangling the ErbB signalling network. Nature
Reviews. Molecular Cell Biology, 2(2), 127–137. http://dx.doi.org/10.1038/35052073.
Zhang, Q. C., Petrey, D., Garzon, J. I., Deng, L., & Honig, B. (2013). PrePPI: A structure-
informed database of protein-protein interactions. Nucleic Acids Research, 41,
D828–D833. http://dx.doi.org/10.1093/nar/gks1231.
Zhu, C., Byers, K. J., McCord, R. P., Shi, Z., Berger, M. F., Newburger, D. E., et al. (2009).
High-resolution DNA-binding specificity analysis of yeast transcription factors. Genome
Research, 19(4), 556–566. http://dx.doi.org/10.1101/gr.090233.108.
CHAPTER NINE
Application of Proteomics
in Diagnosis of ADHD,
Schizophrenia, Major Depression,
and Suicidal Behavior
Khaled Alawam1
Forensic Medicine Department, Ministry of Interior, Kuwait City, Kuwait
1
Corresponding author: e-mail address: [email protected]
Contents
1. Introduction 284
2. Analytical Approaches Applied 286
2.1 Background 286
2.2 Proteomic analysis 286
2.3 Genomic and transcriptomic analysis 287
2.4 Metabolomics 287
3. Analytical Techniques 288
3.1 Protein profiling using matrix-assisted laser desorption-ionization
time-of-flight mass spectrometry 288
3.2 Gas chromatography–mass spectrometry 289
3.3 Fourier transform infrared spectroscopy 290
4. Biomarkers 291
4.1 Background 291
4.2 Types of biomarker 291
5. Depression 292
5.1 Definition of depression 292
5.2 Symptoms 292
5.3 Types of depression 293
5.4 Prevalence of major depression 293
5.5 Major depression theories 294
5.6 Comorbidity of other conditions with major depression 295
5.7 The potential benefit of biomarkers in depression 295
5.8 Forensic aspects of depressive disorders 296
6. Suicidal Behavior 297
6.1 Background 297
6.2 Definition 298
6.3 Risk factors 298
6.4 Prevalence 299
#
Advances in Protein Chemistry and Structural Biology, Volume 95 2014 Elsevier Inc. 283
ISSN 1876-1623 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00009-9
284 Khaled Alawam
Abstract
This report focuses on the application of different proteomic techniques in diagnosis
and treatment of psychiatric disorders such as major depression, suicidal behavior,
schizophrenia, and attention deficit/hyperactivity disorder (ADHD). Firstly, we briefly
describe different analytic approaches that can be applied for the discovery of specific
biomarkers for diagnosing the above disorders, as well as for monitoring the effect of
their treatment. Secondly, we discussed the types of biomarkers in general used in bio-
medicine for characterizing different disorders and diseases. Next, the potential appli-
cations of these biomarkers for diagnosing and managing major depression, suicidal
behavior, schizophrenia, and ADHD are discussed in details. Forensic aspects of these
biomarkers for the above disorders are also considered. Finally, we discuss the potential
of specific biomarkers for distinguishing between comorbid psychiatric disorders in clin-
ical setup as well as their potential for understanding mechanisms underlying the dis-
orders and in discovery of new treatment strategies.
1. INTRODUCTION
The primary focus of this review is the application of proteomic and
metabolomic protocols to the study of serum-based biomarkers of depres-
sion and associated suicidal tendencies. Major depression is a widespread
and devastating psychiatric condition with the majority of cases exhibiting
suicidal behavior after a time suffering from depression. Furthermore, other
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 285
2.4. Metabolomics
Metabolomic analysis encompasses the study of metabolite profiles, which
represent the end products of cellular processes in biological samples.
288 Khaled Alawam
3. ANALYTICAL TECHNIQUES
3.1. Protein profiling using matrix-assisted laser
desorption-ionization time-of-flight mass
spectrometry
Matrix-assisted laser desorption is an ionization method having been
described first by Karas (Costello, 1997; Karas & Hillenkamp, 1988). Their
work demonstrated that this techniques and results obtained can be consid-
ered a revolution in the way that MS experiments allowing the analysis of
biomolecules (biopolymers such as proteins, peptides, and sugars) and large
organic molecules (such as polymers, dendrimers, and other macromole-
cules). Franz Hillenkamp and colleagues by 1987 discovered that ions can
be produced from large proteins by laser desorption if these molecules are
mixed with small organic compounds working as matrices that strongly
absorbance the laser wavelength aimed at them during ionization. The sam-
ples if ionized can be analyzed based on their differing masses and relative
abundance. Matrix-assisted laser desorption-ionization time-of-flight mass
spectrometry (MALDI-TOF MS) has become an important tool in the anal-
ysis of biomolecules.
The MALDI ionization process consists of a number of variables which
are optimized based upon the type of biomolecule being analyzed and sam-
ple source, as discussed below:
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 289
4. BIOMARKERS
4.1. Background
Psychiatric research commonly focuses on genomic pathways to reveal
genes abnormally expressed in patients and their products thus understand-
ing the pathophysiology of different neuropsychiatric disorders. Protein bio-
marker discovery can also be employed in neuropsychiatric disorders in
addition to genetic studies. There is also a growing need to identify various
biomarkers associated with psychiatric disorders to support the theories
regarding the initial development of the disorders and identify new target
sites for therapy development.
Biomarker assessment might be undertaken by studying either bodily
fluid or body tissue as initial sources of biological material. Body fluids avail-
able for study include serum, saliva, urine, and CSF, whereas body tissue
relies on biopsy (which is impractical and dangerous to the patient if at all
reliable in neuropsychiatric disorders), human postmortem samples, or cell
lines. Of these options, biological fluid collection is the easiest to access and
also the least invasive to the patient and serum has been a predominant focus
for previous biomarker studies. Biomarkers may also facilitate the identifi-
cation of treatment effects on disease progression and so aid the therapeutic
development related to the disorder and personalized treatments. Further-
more, specific biomarkers might also be considered not only in diagnostic
criteria but also as an indicator of prognosis.
biomarkers. The trait biomarkers are enduring and their levels reflect the
property of behavior and biological functioning which potentially play a role
in the etiopathology of disorders. Therefore, trait biomarkers can be present
in clinically unaffected individuals prior to any development of the disorder.
Trait biomarkers are representative for the origin of the disorder rather than
its expression in individuals. The state biomarkers are transient and charac-
terize the clinical manifestation of disorders rather than any disposition
toward them. Unlike trait biomarkers, state biomarkers would return to
“normal” levels with successful therapy and so can act as a prognostic tool
(Chen, Bidwell, & Norton, 2006).
5. DEPRESSION
5.1. Definition of depression
Depression is considered in part to be a completely normal stress reaction to a
number of factors that may be experienced by an individual. The depression
is only considered as being abnormal if it is disproportionate to the triggering
event, and when the individuals are determined to be unable to relieve
themselves of the depressive state. Depression is a very common psychiatric
disease worldwide and is estimated to become the second most common
cause of disability, after heart disease by 2020 (Peveler, Carson, & Rodin,
2002). People suffering with depression may be subjected to discrimination
and are more likely to be unemployed compared to other people in the gen-
eral population and other groups with mental disorders. The effect of the
disorder itself and the stigma attached to the disorder leads to a greater poten-
tial for people with depression becoming socially isolated, which in turn
reemphasizes the individuals’ feelings of worthlessness and hopelessness.
Depressive disorder as a psychiatric condition is characterized by psycholog-
ical, behavioral, and physical symptoms (Greden, 2003).
5.2. Symptoms
The symptoms of depression develop over an extended time period and for
the purpose of a clinical diagnosis it is considered that five symptoms should
be present and persist for a period of 2 weeks. Such diagnosis is based upon
patients’ self-reported or relative-reported behavior of the patient. Diagnosis
criteria for depression are detailed by the Diagnostic and Statistical Manual-
fourth edition (DSM-IV) of the American Psychiatric Association and the
International Classification of Diseases and Related Health Problems
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 293
these potential factors among others, it may also be possible that other factors
including genetic, psychological, and environmental factors might also be
involved in the development of the condition.
6. SUICIDAL BEHAVIOR
6.1. Background
Suicidal behavior includes complete suicide, attempted suicide, and suicide
ideation and such behavior may be considered to be an indicator of inade-
quate methods or application of screening of patients by the mental health
services within any specific region. Suicide behavior is always deliberate,
conscious, and represents emotional feelings with a strong passion for strong,
aggressive self-destructive acts. The suicide attempt is generally considered
to be a “call for help” or a way to communicate with others with the aim of
298 Khaled Alawam
changing the behavior of others, the primary motive is not therefore the
death of the individual.
6.2. Definition
The overall definition of suicide has been widely debated and varies
depending upon the mental illness, cultural, and religion. Generally, suicide
is defined by a person intentionally attempting to take his or her own life and
the World Health Organization defines suicide as the act of deliberately kill-
ing oneself. Many mental disorders are considered to be risk factors for sui-
cide behavior; thus, collaboration between psychiatric and social services
may improve suicide prevention strategies, initiating care of the patient
when suicidal thoughts first occur. Although suicide is a serious problem
for patients with psychiatric distress, especially for those with depression,
it is also considered as a component of the psychiatric symptoms and may
be related to other disorders associated with depression and therefore can
be easily misdiagnosed. For example, suicidal behavior may also be a com-
ponent of disorders such as anxiety and alcohol abuse. Generally, individuals
who make serious suicide attempts have high rates of mental disorders
(Beautrais et al., 1996); therefore, suicide is considered to the misdiagnosis
of a mental disorder. Improving physician education in depression disorder
recognition and treatment and restricting access of those with such risk fac-
tors to lethal methods has been shown to be a positive method of reducing
suicide rates (Mann et al., 2005).
6.4. Prevalence
Among ethnic groups suicide behavior is more recognized in association
with depression (Oquendo et al., 2004). Several reasons for the prevalence
of suicide behavior need to be considered as it is recognized as a multicausal
behavior with biological factors, psychopathological factors, and social and
cultural environment all playing important roles in the determination and
manifestation of suicidal behavior. Results from a study in Denmark have
shown that, across the entire population, prevalence of suicidal ideation is
6.9% and of suicidal attempts 3.4% (Kjøller & Helweg-Larsen, 2000). Being
female is associated with a higher prevalence of suicide ideation and attempts
by females who are divorced or widowed at a young age show a further
increase in prevalence. Although, there were significant differences in gen-
der as a Swedish study showed a higher suicide rate toward men (71%) com-
pared to women (29%) (Holmgren & Jones, 2010). Good mental health
services might increase life expectancy because females with major
300 Khaled Alawam
6.5. Comorbidity
As mentioned previously, among cases of suicide, approximately 90% of
individuals had one or more psychiatric disorders at the time they committed
suicide (Arsenault-Lapierre et al., 2004). The most prevalent disorders were
depressive syndromes (66%) and alcohol dependence/abuse (43%)
(L€
onnqvist et al., 1995). The study of suicide victims from the nationwide
“suicide population” showed that at least one psychiatric diagnosis was pre-
sent in 93% of the victims. Other results among the subjects who had com-
mitted suicide demonstrated that 38.7% were afflicted by major depression,
24% by alcohol dependency, and 28.7% were dependent on drugs
(Grunberg et al., 1994). Therefore, mental disorders have been found in
most people making serious suicide attempts with suicide risk being partic-
ularly high in those with unobserved suicide intent, depression patients,
patients with chronic alcohol and drug misuse, social isolation, and current
physical illnesses (Mitchell & Dennis, 2006).
recorded and shared for future use. The health records in forensic psychiatry
for reporting the clinical evidence regarding suicide victims are based on an
interviewed assessment which might not be sufficient, or even possible, in
some cases and so adding objective biomarkers may assist in helping reach a
forensic decision. Furthermore, biomarkers may exhibit a predictive value
and serve as screening tools in the prevention program.
7. SCHIZOPHRENIA
7.1. Background
Schizophrenia is a psychiatric disorder characterized by disruption in cog-
nition and emotion which affects many patients’ attributes including
thoughts, perception, and speech. Diagnosis of schizophrenia is usually
made with reference to the criteria in DSM-IV and ICD-10 which states
that for a definitive diagnosis at least 1 month duration of two or more pos-
itive symptoms which reflect a function distortion should be identified.
The negative symptoms of schizophrenia are more difficult to evaluate
and persist in schizophrenic patients during periods in which positive
symptoms are absent. The disorder has several neurobiological abnormal-
ities with a previous etiology study suggesting that the disorder developed
due to a gene–environment interaction (van Os, Rutten, & Poulton,
2008). Biological abnormalities of the disorder are mostly detected
through image-based biomarkers such as abnormally large brain ventricles
due to CSF, decrease in grey matter, and alteration of white matter
(Davatzikos et al., 2005). However, the study of the dopamine system
has demonstrated abnormal dopamine concentrations, especially in the
acute psychotic state of the disorder, which has represented a major focus
of schizophrenia research (Howes & Kapur, 2009). Moreover, other stud-
ies have focused on the increase in dopamine synthesis, dopamine release,
and resting-state synaptic dopamine levels. This too has dictated much
research in respect of schizophrenia treatment strategy, with most treat-
ments mediating the blocking of dopamine receptors especially in the psy-
chosis state (Kapur, Agid, Mizrahi, & Li, 2006). In depression patients
with schizophrenia, the SSRI treatments are the most effective therapy
(Kasckow, Felmet, & Zisook, 2011). Gender variation also has been
suggested to have a role as the disorder is significantly more common in
males (Castle, Wessely, & Murray, 1993; McGrath, Saha, Chant, &
Welham, 2008).
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 303
7.2. Symptoms
The recognized symptoms of schizophrenia are listed by DSM-IV and are
grouped into three groupings. Positive symptoms commonly include delu-
sion (false beliefs), hallucination (including visual, auditory or tactile, olfac-
tory, and gustatory hallucinations), disorganized speech, and disorganized
behavior (catatonic behavior, abnormal and/or extreme rigidity/flexibility
of limbs). Negative symptoms include alogia (lack of additional normal
speech), avolition (lack of motivation), affective flattening (lack of emotion),
and finally, cognitive symptoms such as slow/disorganized thinking, poor
concentration, poor memory, difficulty in understanding, and abnormal
feeling behavior can be observed.
symptoms which result in a higher rate of death via suicide and suicide
behavior compared to patients with schizophrenia alone (Caldwell &
Gottesman, 1990; Inskip, Harris, & Barraclough, 1998; Isjanovski,
Vukovic, Hadjihamza, Pejoska-Gerazova, & Raleva, 2010; Kim et al.,
2010; Palmer, Pankratz, & Bostwick, 2005; Saha, Chant, & McGrath,
2007). Therefore, suicide victims may encompass a wide range of underlying
mental disorders; moreover, transient depressive mood pictures represent an
increased risk stage for suicide behavior (Fenton, 2000; Hawton, Sutton,
Haw, Sinclair, & Deeks, 2005).
Other misdiagnosed disorders, such as adult ADHD, are also associated
with criminal behavior (de Andrade et al., 2011) and thought to underline
any schizophrenia disorder present. Therefore, forensic examiners are
required to improve their awareness in relation to undiagnosed disorders
leading to persistence of antisocial behavior if not referred and treated by
expert. Moreover, screening at custody services may have potential value
in predicting other family members who may be at risk of not being diag-
nosed as also having symptoms, misdiagnosis, and the impact of this on
potential future criminal behavior.
might reveal the etiology of disorder. Furthermore, social factors and arti-
ficial food additives have also been considered as contributing to the etiology
of ADHD (McCann et al., 2007). Such research has the potential to identify
positive family history factors and help tracing trait biomarkers which under-
line the disorder. The pathophysiology of ADHD is unclear, however,
imaging analysis has previously shown several abnormalities of brain volume
in childhood patients with a total reduction of cortical and cerebral areas
being reported alongside a decrease in cortical folding (Wolosin,
Richardson, Hennessey, Denckla, & Mostofsky, 2009). There is also some
evidence that the dopaminergic neurotransmitter system may be involved in
the pathophysiology of ADHD (Tripp & Wickens, 2009). Additionally, a
plethora of other factors such as neurotrophins and CLOCK genes have
been implicated in the path-cascade ultimately leading to ADHD
(Conner et al., 2008; Kent et al., 2005; Kissling et al., 2008).
8.2. Symptoms
ADHD symptoms are listed in the DSM-IV and International Statistical
Classification of Disease-10 Revision (ICD-10) with core symptoms being
evident in early childhood. These symptoms are predominantly: increased
levels of hyperactivity, impulsivity, and inattention. Adult ADHD patients
typically also exhibit these same tendencies (Wender, Wolf, &
Wasserstein, 2001).
8.4. Comorbidity
ADHD may be associated with other disorders which can complicate the
diagnosis and treatment of the patient. The most commonly identified
comorbid disorders are conduct disorder, mood disorder, depression, anx-
iety disorder, and learning disability disorder (Biederman, Newcorn, &
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 307
9. CONCLUDING REMARKS
The psychiatry service has a long way to go to develop standard criteria
based on laboratory-derived evidence, alongside the usually performed psy-
chometric results which are considered as a psychiatric (although potentially
subjective) opinion concerning the mental health of individuals. Moreover,
the development of a linkage between biomarker evidence and different
psychometric tools would be envisaged to optimize such psychiatric-
standard screening, diagnosis, and management strategies. Therefore, the
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 309
REFERENCES
Ackerman, M. J. (2006). Forensic report writing. Journal of Clinical Psychology, 62, 59–72.
Amico, F., Meisenzahl, E., Koutsouleris, N., Reiser, M., M€ oller, H. J., & Frodl, T. (2011).
Structural MRI correlates for vulnerability and resilience to major depressive disorder.
Psychiatry and Neuroscience, 36, 15–22.
Arango, V., Underwood, M. D., & Mann, J. J. (1996). Fewer pigmented locus coeruleus
neurons in suicide victims: Preliminary results. Biological Psychiatry, 39, 112–120.
Arsenault-Lapierre, G., Kim, C., & Turecki, G. (2004). Psychiatric diagnosis in 3275 sui-
cides: A meta-analysis. BMC Psychiatry, 4, 37.
Baillargeon, J., Penn, J. V., Thomas, C. R., Temple, J. R., Baillargeon, G., & Murray, O. J.
(2009). Psychiatric disorders and suicide in the nation’s largest state prison system. Journal
of the American Academy of Psychiatry and the Law, 37, 188–193.
Barkley, R. A., Fischer, M., Smallish, L., & Fletcher, K. (2002). The persistence of attention-
deficit/hyperactivity disorder into young adulthood as a function of reporting source and
definition of disorder. Journal of Abnormal Psychology, 111, 279–289.
Beautrais, A. L., Joyce, P. R., Mulder, R. T., Fergusson, D. M., Deavoll, B. J., &
Nightingale, S. K. (1996). Prevalence and co-morbidity of mental disorders in persons
making serious suicide attempts: A case control study. American Journal of Psychiatry, 153,
1009–1014.
Bellak, L., Kay, S. R., & Opler, L. A. (1987). Attention deficit disorder psychosis as a diag-
nostic category. Psychiatric Developments, 5, 239–263.
310 Khaled Alawam
Bernal, M., Haro, J. M., Bernert, S., Brugha, T., de Graaf, R., Bruffaerts, R., et al. (2007).
Risk factors for suicidality in Europe: Results from the ESEMED study. Journal of Affective
Disorders, 101, 27–34.
Biederman, J., Newcorn, J., & Sprich, S. (1991). Comorbidity of attention deficit hyperac-
tivity disorder with conduct, depressive, anxiety, and other disorders. American Journal of
Psychiatry, 148, 564–577.
Bondy, B., Buettner, A., & Zill, P. (2006). Genetics of suicide. Molecular Psychiatry, 11,
336–351.
Breslau, N., Davis, G. C., Peterson, E. L., & Schultz, L. R. (2000). A second look at comor-
bidity in victims of trauma: The posttraumatic stress disorder-major depression connec-
tion. Biological Psychiatry, 48, 902–909.
Caldwell, C. B., & Gottesman, I. I. (1990). Schizophrenics kill themselves too; our view of
risk factors for suicide. Schizophrenia Bulletin, 16, 571–590.
Caspi, A., Langley, K., Milne, B., Moffitt, T. E., O’Donovan, M., Owen, M. J., et al. (2008).
A replicated molecular genetic basis for subtyping antisocial behavior in children with
attention-deficit/hyperactivity disorder. Archives of General Psychiatry, 65, 203–210.
Castle, D. J., Wessely, S., & Murray, R. M. (1993). Sex and schizophrenia: Effects of diag-
nostic stringency, and associations with and premorbid variables. British Journal of Psychi-
atry, 162, 658–664.
Centers for Disease Control and Prevention (CDC). (2006). Homicides and suicides—
National Violent Death Reporting System, United States, 2003–2004. Morbidity and
Mortality Weekly Report, 55, 721–724.
Chen, Y., Bidwell, L. C., & Norton, D. (2006). Trait vs. state markers for schizophrenia:
Identification and characterization through visual processes. Current Psychiatry Reviews,
2, 431–438.
Cohen, M., Yossef, R., Erez, T., Kugel, A., Welt, M., Karpasas, M. M., et al. (2011). Serum
apolipoproteins C-I and C-III are reduced in stomach cancer patients: Results from
MALDI-based peptidome and immuno-based clinical assays. PLoS One, 6, e14540.
Conner, A. C., Kissling, C., Hodges, E., Hünnerkopf, R., Clement, R. M., Dudley, E., et al.
(2008). Neurotrophic factor-related gene polymorphisms and adult attention deficit
hyperactivity disorder (ADHD) score in a high-risk male population. American Journal
of Medical Genetics Part B: Neuropsychiatric Genetics, 147B, 1476–1480.
Coppen, A., & Bolander-Gouaille, C. (2005). Treatment of depression: Time to consider
folic acid and vitamin B12. Journal of Psychopharmacology, 19, 59–65.
Corbett, B. A., Kantor, A. B., Schulman, H., Walker, W. L., Lit, L., Ashwood, P., et al. (2007).
A proteomic study of serum from children with autism showing differential expression of
apolipoproteins and complement proteins. Molecular Psychiatry, 12, 292–306.
Costello, C. E. (1997). Time, life. . . and mass spectrometry. New techniques to address bio-
logical questions. Biophysical Chemistry, 68, 173–188.
Dailly, E., Chenu, F., Renard, C. E., & Bourin, M. (2004). Dopamine, depression and anti-
depressants. Fundamental & Clinical Pharmacology, 18, 601–607.
Davatzikos, C., Shen, D., Gur, R. C., Wu, X., Liu, D., Fan, Y., et al. (2005). Whole brain
morphometric study of schizophrenia revealing a spatially complex set of focal abnormal-
ities. Archives of General Psychiatry, 62, 1218–1227.
Dawson, D. A., Grant, B. F., Stinson, F. S., & Chou, P. S. (2005). Psychopathology asso-
ciated with drinking and alcohol use disorders in the college and general adult
populations. Drug and Alcohol Dependence, 77, 139–150.
de Andrade, R. C., Assumpção, F., Jr., & Teixeira, I. A. (2011). Prevalence of psychiatric
disorders in juvenile offenders in the city of Rio de Janeiro (RJ, Brazil). Ciência & Saúde
Coletiva, 16, 2179–2188.
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 311
Dudley, E., Hässler, F., & Thome, J. (2011). Profiling for novel proteomics biomarkers in
neurodevelopmental disorders. Expert Review of Proteomics, 8, 127–136.
Duman, R. S., Malberg, J., & Thome, J. (1999). Neural plasticity to stress and antidepressant
treatment. Biological Psychiatry, 46, 1181–1191.
Fenton, W. S. (2000). Depression, suicide, and suicide prevention in schizophrenia. Suicide &
Life-Threatening Behavior, 30, 34–49.
Fredericks, J. D., Bennett, P., Williams, A., & Rogers, K. D. (2012). FTIR spectroscopy:
A new diagnostic tool to aid DNA analysis from heated bone. Forensic Science International.
Genetics, 6, 375–380.
Graz, C., Etschel, E., Schoech, H., & Soyka, M. (2009). Criminal behaviour and violent
crimes in former inpatients with affective disorder. Journal of Affective Disorders, 117,
98–103.
Greden, J. F. (2003). Physical symptoms of depression: Unmet needs. Journal of Clinical Psy-
chiatry, 64, 5–11.
Grunberg, F., Lesage, A. D., Boyer, R., Vanier, C., Morissette, R., Buteau, C. M., et al.
(1994). Suicide among young male adults in Quebec: Psychopathology and utilization
of medical services. Santé Mentale au Québec, 19, 25–39.
Halbach, O. B. (2010). Involvement of BDNF in age-dependent alterations in the hippo-
campus. Frontiers in Aging Neuroscience, 13, 36.
Hawton, K., Sutton, L., Haw, C., Sinclair, J., & Deeks, J. J. (2005). Schizophrenia and sui-
cide: Systematic review of risk factors. British Journal of Psychiatry, 187, 9–20.
Hirschfeld, R. M. (2000). History and evolution of monoamine hypothesis of depression.
Journal of Clinical Psychiatry, 61, 4–6.
Hirschfeld, R. M. (2001). The comorbidity of major depression and anxiety disorders: Rec-
ognition and management in primary care. Primary Care Companion to the Journal of Clin-
ical Psychiatry, 3, 244–254.
Holmgren, A., & Jones, A. W. (2010). Demographics of suicide victims in Sweden in relation
to their blood-alcohol concentration and the circumstances and manner of death. Forensic
Science International, 198, 17–22.
Howes, O. D., & Kapur, S. (2009). The dopamine hypothesis of schizophrenia: Version III—
The final common pathway. Schizophrenia Bulletin, 35, 549–562.
Husain, M. M., Rush, A. J., Trivedi, M. H., McClintock, S. M., Wisniewski, S. R.,
Davis, L., et al. (2007). Pain in depression: STAR*D study findings. Journal of Psychoso-
matic Research, 63, 113–122.
Inskip, H. M., Harris, E. C., & Barraclough, B. (1998). Lifetime risk of suicide for affective
disorder, alcoholism and schizophrenia. British Journal of Psychiatry, 172, 35–37.
Isacsson, G., Holmgren, P., Wasserman, D., & Bergman, U. (1994). Use of antidepressants
among people committing suicide in Sweden. British Medical Journal, 308, 506–509.
Isjanovski, V., Vukovic, V., Hadjihamza, K., Pejoska-Gerazova, V., & Raleva, M. (2010).
Some characteristics of patients with schizophrenia who attempted suicide. Prilozi, 31,
183–193.
Iwasaki, M., Akechi, T., Uchitomi, Y., & Tsugane, S. (2005). Cigarette smoking and com-
pleted suicide among middle-aged men: A population-based cohort study in Japan.
Annals of Epidemiology, 15, 286–292.
Jones, R. M., Hales, H., Butwell, M., Ferriter, M., & Taylor, P. J. (2010). Suicide in high
security hospital patients. Social Psychiatry and Psychiatric Epidemiology, 46, 723–731.
Kapur, S., Agid, O., Mizrahi, R., & Li, M. (2006). How antipsychotics work-from receptors
to reality. NeuroRx, 3, 10–21.
Karas, M., & Hillenkamp, F. (1988). Laser desorption ionization of proteins with molecular
masses exceeding 10,000 daltons. Analytical Chemistry, 60, 2299–2301.
312 Khaled Alawam
Karch, C. M., Jeng, A. T., Nowotny, P., Cady, J., Cruchaga, C., & Goate, A. M. (2012).
Expression of novel Alzheimer’s disease risk genes in control and Alzheimer’s disease
brains. PLoS One, 7, e 50976.
Karvonen, K., Hakko, H., Koponen, H., Meyer-Rochow, V. B., & Räsänen, P. (2009). Sui-
cides among older persons in Finland and time since hospitalization discharge. Psychiatric
Services, 60, 390–393.
Kasckow, J., Felmet, K., & Zisook, S. (2011). Managing suicide risk in patients with schizo-
phrenia. CNS Drugs, 25, 129–143.
Kent, L., Green, E., Hawi, Z., Kirley, A., Dudbridge, F., Lowe, N., et al. (2005). Association
of the paternally transmitted copy of common Valine allele of the Val66Met polymor-
phism of the brain-derived neurotrophic factor (BDNF) gene with susceptibility to
ADHD. Molecular Psychiatry, 10, 939–943.
Keshavan, M. S., Sujata, M., Mehra, A., Montrose, D. M., & Sweeney, J. A. (2003). Psy-
chosis proneness and ADHD in young relatives of schizophrenia patients. Schizophrenia
Research, 59, 85–92.
Kessler, R. C., Adler, L., Barkley, R., Biederman, J., Conners, C. K., Demler, O., et al.
(2006). The prevalence and correlates of adult ADHD in the United States: Results from
the National Comorbidity Survey Replication. American Journal of Psychiatry, 163,
716–723.
Kim, S. W., Kim, S. J., Mun, J. W., Bae, K. Y., Kim, J. M., Kim, S. Y., et al. (2010). Psy-
chosocial factors contributing to suicidal ideation in hospitalized schizophrenia patients
in Korea. Psychiatry Investigation, 7, 79–85.
Kissling, C., Retz, W., Wiemann, S., Coogan, A. N., Clement, R. M., Hünnerkopf, R.,
et al. (2008). A polymorphism at the 3’-untranslated region of the CLOCK gene is asso-
ciated with adult attention-deficit hyperactivity disorder. American Journal of Medical
Genetics Part B: Neuropsychiatric Genetics, 147, 333–338.
Kjøller, M., & Helweg-Larsen, M. (2000). Suicidal ideation and suicide attempts among adult
Danes. Scandinavian Journal of Public Health, 28, 54–61.
Kuehner, C. (2003). Gender differences in unipolar depression: An update of epidemiolog-
ical findings and possible explanations. Acta Psychiatrica Scandinavica, 108, 163–174.
LaFrance, W. C., Jr., Kanner, A. M., & Hermann, B. (2008). Psychiatric comorbidities in
epilepsy. International Review of Neurobiology, 83, 347–383.
Langley, K., Fowler, T., Ford, T., Thapar, A. K., van den Bree, M., Harold, G., et al. (2010).
Adolescent clinical outcomes for young people with attention-deficit hyperactivity dis-
order. British Journal of Psychiatry, 196, 235–240.
Li, Y. H., Wang, J., Zheng, X. L., Zhang, Y. L., Li, X., Yu, S., et al. (2011). Matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry combined with magnetic
beads for detecting serum protein biomarkers in Parkinson’s disease. European Neurology,
65, 105–111.
Liu, C., Pan, C., Shen, J., Wang, H., & Yong, L. (2011). MALDI-TOF MS combined with
magnetic beads for detecting serum protein biomarkers and establishment of boosting
decision tree model for diagnosis of colorectal cancer. International Journal of Medical
Sciences, 8, 39–47.
L€
onnqvist, J. K., Henriksson, M. M., Isometsä, E. T., Marttunen, M. J., Heikkinen, M. E.,
Aro, H. M., et al. (1995). Mental disorders and suicide prevention. Psychiatry and Clinical
Neurosciences, 49, S111–S116.
Lopez, M. F., Mikulskis, A., Kuzdzal, S., Bennett, D. A., Kelly, J., Golenko, E., et al. (2005).
High-resolution serum proteomic profiling of Alzheimer disease samples reveals disease-
specific, carrier-protein-bound mass signatures. Clinical Chemistry, 51, 1946–1954.
MacDonald, R., Taylor, J., & Clarke, D. (2009). The relationship between early suicide
behaviors and mental health: Results from a nine-year panel study. Journal of Adolescence,
32, 1159–1172.
Proteomics in ADHD, Schizophrenia, Depression, and Suicide 313
Mann, J. J., Apter, A., Bertolote, J., Beautrais, A., Currier, D., Haas, A., et al. (2005). Suicide
prevention strategies: A systematic review. JAMA, 294, 2064–2074.
Mannuzza, S., Klein, R. G., & Moulton, J. L., 3rd. (2008). Lifetime criminality among boys
with attention deficit hyperactivity disorder: A prospective follow-up study into adult-
hood using official arrest records. Psychiatry Research, 160, 237–246.
Mayes, R., Bagwell, C., & Erkulwater, J. (2008). ADHD and the rise in stimulant use among
children. Harvard Review of Psychiatry, 16, 151–166.
McCann, D., Barrett, A., Cooper, A., Crumpler, D., Dalen, L., Grimshaw, K., et al. (2007).
Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the
community: A randomised, double-blinded, placebo-controlled trial. Lancet, 370,
1560–1567.
McGrath, J., Saha, S., Chant, D., & Welham, J. (2008). Schizophrenia: A concise overview of
incidence, prevalence, and mortality. Epidemiologic Reviews, 30, 67–76.
McIntosh, D., Kutcher, S., Binder, C., Levitt, A., Fallu, A., & Rosenbluth, M. (2009). Adult
ADHD and comorbid depression: A consensus-derived diagnostic algorithm for ADHD.
Neuropsychiatric Disease and Treatment, 5, 137–150.
Mekonnen, D., & Kebede, Y. (2011). The prevalence of suicidal ideation and attempts
among individuals attending an adult psychiatry out-patient clinic in Gondar, Ethiopia.
African Health Sciences, 11, 103–107.
Meyer, J. H., Ginovart, N., Boovariwala, A., Sagrati, S., Hussey, D., Garcia, A., et al. (2006).
Elevated monoamine oxidase a levels in the brain: An explanation for the monoamine
imbalance of major depression. Archives of General Psychiatry, 63, 1209–1216.
Mitchell, A. J., & Dennis, M. (2006). Self-harm and attempted suicide in adults: 10 practical
questions and answers for emergency department staff. Emergency Medicine Journal, 23,
251–255.
Müller, U. C., Asherson, P., Banaschewski, T., Buitelaar, J. K., Ebstein, R. P., Eisenberg, J.,
et al. (2011). The impact of study design and diagnostic approach in a large multi-centre
ADHD study. Part 1: ADHD symptom patterns. BMC Psychiatry, 11, 54.
Oquendo, M. A., Lizardi, D., Greenwald, S., Weissman, M. M., & Mann, J. J. (2004). Rates
of lifetime suicide attempt and rates of lifetime major depression in different ethnic
groups in the United States. Acta Psychiatrica Scandinavica, 110, 446–451.
Palmer, B. A., Pankratz, V. S., & Bostwick, J. M. (2005). The lifetime risk of suicide in
schizophrenia: A reexamination. Archives of General Psychiatry, 62, 247–253.
Pandey, S., Pandey, P., Tiwari, G., Tiwari, R., & Rai, A. K. (2012). FTIR spectroscopy:
A tool for quantitative analysis of ciprofloxacin in tablets. Indian Journal of Pharmaceutical
Sciences, 74, 86–90.
Peveler, R., Carson, A., & Rodin, G. (2002). ABC of psychological medicine. Depression in
medical patients. British Medical Journal, 325, 149–152.
Powell, J., Geddes, J., Deeks, J., Goldacre, M., & Hawton, K. (2000). Suicide in psychiatric
hospital in-patients. Risk factors and their predictive power. British Journal of Psychiatry,
176, 266–272.
Qin, P., Agerbo, E., & Mortensen, P. B. (2003). Suicide risk in relation to socioeconomic,
demographic, psychiatric, and familial factors: A national register-based study of all
suicides in Denmark, 1981–1997. American Journal of Psychiatry, 160, 765–772.
Rathi, Y., Malcolm, J., Michailovich, O., Goldstein, J., Seidman, L., McCarley, R. W., et al.
(2010). Biomarkers for identifying first-episode schizophrenia patients using diffusion
weighted imaging. Medical Image Computing and Computer-Assisted Intervention, 13,
657–665.
Reichert, C. L., Diogo, C. L., Vieira, J. L., & Dalacorte, R. R. (2011). Physical activity and
depressive symptoms in community-dwelling elders from southern Brazil. Revista
Brasileira de Psiquiatria, 33, 165–170.
Rihmer, Z. (2007). Suicide risk in mood disorders. Current Opinion in Psychiatry, 20, 17–22.
314 Khaled Alawam
Wang, D. C., Sun, C. H., Liu, L. Y., Sun, X. H., Jin, X. W., Song, W. L., et al. (2012).
Serum fatty acid profiles using GC-MS and multivariate statistical analysis: Potential bio-
markers of Alzheimer’s disease. Neurobiology of Aging, 33, 1057–1066.
Way, B. B., Miraglia, R., Sawyer, D. A., Beer, R., & Eddy, J. (2005). Factors related to sui-
cide in New York state prisons. International Journal of Law and Psychiatry, 28, 207–221.
Wender, P. H., Wolf, L. E., & Wasserstein, J. (2001). Adults with ADHD. An overview.
Annals of the New York Academy of Sciences, 931, 1–16.
Wolf, L. E., & Wasserstein, J. (2001). Adult ADHD. Concluding thoughts. Annals of the New
York Academy of Sciences, 931, 396–408.
Wolosin, S. M., Richardson, M. E., Hennessey, J. G., Denckla, M. B., & Mostofsky, S. H.
(2009). Abnormal cerebral cortex structure in children with ADHD. Human Brain Map-
ping, 30, 175–184.
Yee, N. Y., Large, M. M., Kemp, R. I., & Nielssen, O. B. (2011). Severe non-lethal violence
during psychotic illness. The Australian and New Zealand Journal of Psychiatry, 45, 466–472.
Yoshimasu, K., Sugahara, H., Tokunaga, S., Akamine, M., Kondo, T., Fujisawa, K., et al.
(2006). Gender differences in psychiatric symptoms related to suicidal ideation in
Japanese patients with depression. Psychiatry and Clinical Neurosciences, 60, 563–569.
Young, S. J., Adamou, M., Bolea, B., Gudjonsson, G., Müller, U., Pitts, M., et al. (2011).
The identification and management of ADHD offenders within the criminal justice sys-
tem: A consensus statement from the UK Adult ADHD Network and criminal justice
agencies. BMC Psychiatry, 11, 32.
Young, S., Gudjonsson, G. H., Wells, J., Asherson, P., Theobald, D., Oliver, B., et al.
(2009). Attention deficit hyperactivity disorder and critical incidents in a Scottish prison
population. Personality and Individual Differences, 46, 265–269.
Zhao, L., Liu, Y., Sun, X., Peng, K., & Ding, Y. (2011). Serum proteome analysis for pro-
filing protein markers associated with lymph node metastasis in colorectal carcinoma.
Journal of Comparative Pathology, 144, 187–194.
AUTHOR INDEX
Note: Page numbers followed by “f ” indicate figures, “t ” indicate tables and “np” indicate
footnotes.
317
318 Author Index
Barber, N., 248 Belmonte, L., 138–140, 141, 142, 164, 165,
Barbieri, F., 182–183 166–168, 170, 171–172, 177
Barbosa, A. E., 15–16 Belov, L., 248
Barboza, M., 73 Bencharit, S., 138
Barbuti, S., 131, 136–137, 148 Bennett, D. A., 286
Barington, T., 220, 221t Bennett, P., 291
Bar-Joseph, Z., 135 Ben-Nissan, B., 126
Barker, K., 140 Benschop, J. J., 35–36
Barkley, R. A., 305–307 Benson, D. A., 135
Barnes, K. C., 135 Beranova-Giorgianni, S., 41, 48–49
Barone, A., 126, 131, 136–137, Berger, M. F., 235–236, 255, 256–257, 258
148 Bergeron, J. J., 226–227
Barone, R., 104–105, 106 Bergman, U., 300–301
Barquero, M.-S., 104–105 Berjanskii, M., 204
Barraclough, B., 304–305 Berman, A., 172–173
Barrasa, M. I., 258–259 Bern, M., 90–92
Barrett, A., 305–306 Berna, M., 4
Barrios-Rodiles, M., 234 Bernal, M., 299–300
Barshir, R., 262t Bernert, S., 299–300
Barthelery, M., 221t, 222–223 Bernhard, F., 252–253
Barton, G. J., 262t Bernhardt, J., 11
Bartos, A., 3–4 Bernstorff, S., 172–173
Basha, O., 262t Berntenis, N., 3–4
Basrur, V., 45–48 Bertl, K., 128
Bass, M., 172–173 Bertleff, N., 12
Basse, M. J., 262t Bertolote, J., 298
Bassim, C. W., 130 Bertolusso, G., 149
Bauer, G., 172–173 Bertozzi, C. R., 72–73
Bausch-Fluck, D., 220, 221t Berven, F. S., 79–80
Bavastrello, V., 126, 139–140, 141, 143, Berweger, S., 165
167–168, 185 Beseme, O., 55–58
Baxter, S. S., 138 Betesh, L. R., 82–83
Baynes, J. W., 81 Beveridge, A. J., 106
Beare, P. A., 249–250 Bezerra, T., 167–168, 185
Beausoleil, S. A., 37, 39–40, 42–43 Bharti, B., 128
Beautrais, A., 298 Bhattacharya, S., 220, 221t
Beautrais, A. L., 298 Bhola, N. E., 50–51
Bechard, M., 217 Bhongade, M., 128
Becker, K. G., 135 Bhullar, B., 140, 142–143, 238t, 243–244
Bedi, G., 6 Bi, C., 76–77
Beer, L. A., 3 Bian, X., 140
Beer, R., 298–299 Bibel, M., 220, 221t
Behrendt, K., 45–48 Bichsel, V. E., 238t
Bell, A. W., 226–227 Bidwell, L. C., 291–292
Bellak, L., 307 Biederman, J., 305–307
Belli, H., 304–305 Biegler, J. M., 181–182
Belliard, A., 55–58 Biggart, E., 250
Belloni, M., 45–48 Bilello, J., 287–288
320 Author Index
Spera, R., 126, 138–140, 141, 142, 143, Strauss, J. F., 181–182
144, 145, 164–165, 166–168, 170, Strimbu, K., 126–127
171–173, 180–181, 182, 184, 185 Strum, J. S., 85, 87
Spirin, S., 262t Struwe, W. B., 96–98, 103
Spotorno, L., 126 Stukenberg, P. T., 37
Sprenger, J., 226 Stumpf, M. P., 235t
Sprich, S., 306–307 Stura, E., 139–140, 141, 142, 167–168, 170,
Spurrier, B., 254 171–172, 182–183
Sresanga, K., 54–55 Sturiale, L., 104–105, 106
Srikanth, S. M., 151 Sturm, M., 35, 42
Srivastava, S., 139–140, 238t Su, X. D., 147
Srnec, M., 261–269 Subra, G., 204
St John, M. A., 151 Sud, S., 50–51
Stafford, G. P., 149 Sugahara, H., 298–299
Ståhl, S., 48 Sugawara, H., 135
Stanca, S. E., 10 Sugino, H., 220, 221t
Stanley, A. J., 4 Sugiyama, N., 34–35
Stark, C., 262t Sui, S., 40
Steen, H., 7–8, 32–33, 44–45, 98–99 Sujata, M., 307
Stefan, C. J., 220–222 Sultana, R., 104–105
Stefansson, C. G., 301–302 Sun, C. H., 289–290
Steger, G. G., 249 Sun, D., 82–83
Stein, A., 262t Sun, H., 129
Stein, T. I., 135 Sun, J., 261–269
Steinacker, P., 106 Sun, S., 101–102
Steinberg, J., 235–236 Sun, T., 234
Steiner, B., 249 Sun, X., 286
Steinlein, P., 219 Sun, X. H., 289–290
Stelder, S. K., 205 Sun, X.-L., 81
Stellberger, T., 234, 262t Sun, Y., 81
Stelzer, G., 135 Sun, Z., 98–99, 226
Stelzl, U., 235t, 262t Sundar, N. M., 128
Stemman, O., 8 Sung, H.-J., 103
Stene, T., 38 Sung, T. Y., 54
Stevens, R. C., 164–165 Surinova, S., 98–99
Stewart, P. D., 184 Suriyamongkol, B. P., 204
Stewart, R., 235t Surmann, K., 10, 11
Stingl, C., 34, 104–105 Surolia, A., 78, 79
Stinson, F. S., 295 Susin, C., 128
Stoeckert, C. J. Jr., 135 Sutton, L., 304–305
Stoeva, S. I., 250–252 Su, W. C., 82–83, 86–87
Stoevesandt, O., 234 Suzuki, H., 258–259
Stojanov, T., 220, 221t Suzuki, S., 54–55
Stokes, D. L., 164–165 Suzuki, T., 49–50
Stokes, S. T., 102 Suzuki, Y., 34
Storts, D. R., 252 Swaney, D. L., 32–33
Stoyanova, T., 50–51 Swann, J., 15–16
Stoyanovich, J., 135 Swatkoski, S., 130
352 Author Index
A Bioinformatics
Absolute quantification (AQUA) method, 8 glycoproteomic profiling, 98–99
Acinetobacter baumannii, 11–12 leader-gene algorithm, 131–138
Adenosine triphosphate (ATP), 26–28 nanocrystallography, 177–178
ADHD. See Attention deficit/hyperactivity resources, 131
disorder (ADHD) Biomarkers
ADP-ribosylation, 247–248 ADHD, 307
Animal fish model, 13–14 blood-derived, 128
Anodic porous alumina (APA), 141–142 body fluids, 291
Antibody-based strategy, 81–82 cancer research (see Cancer research)
Attention deficit/hyperactivity disorder depression, 295–296
(ADHD) discovery and drug design, 2–3
biomarkers and, 307 schizophrenia, 303–304
comorbidity, 306–307 suicidal behavior, 300–301
forensic aspects, 307–308 types, 291–292
neurodevelopmental disorder, 305–306 Boronic acid chemistry, 81
psychiatric disorders, 307–308 Brucella abortus, 15
subtypes, 306
symptoms, 306 C
Auto-antibodies detection, 249 Cancer research
aberrant hyperfucosylation, 103
B alpha-fetoprotein, 99–101
Bacterial kinases, 253–254 anaplastic lymphoma kinase, 45–48
Bacterial pathogens B-cell lymphoma cell lines, 99–101, 101f
Acinetobacter baumannii, 11–12 castration-resistant metastatic prostate
antibiotic-resistant proteins, 11–12 cancer, 50–51
azidonorleucine, 9–10 collagen receptors, 48
bacterial infection prevention, 12 dasatinib, 52–53
mammal cells, 12 docetaxel resistance, 49–50
multidrug-resistant strains, 11 epidermal growth factor receptor, 45–48
PilE, 10 extracted compound chromatograms,
Pseudomonas aeruginosa, 12 99–101, 100f
secretomics, 9–10 gene silencing technology, 50–51
shotgun proteomics method, Helix pomotia agglutinin lectin,
11–12 102–103
Staphylococcus aureus, 10 imatinib, 51–52
Yersinia enterocolitica type III secretion kinase enzymes, 52–53
system, 9–10 lectins, 101–102
Bacteriorhodopsin, 182–183 liver cirrhosis, 101–102
Bait protein, 234 mucins and glycoproteins, 102–103
Basic helix–loop–helix (bHLH) proteins, phosphoprotein analysis, 45–48
258–259 phosphorylated proteins, 50
359
360 Subject Index