Target identification using drug affinity responsive

target stability (DARTS)

Brett Lomenicka, Rui Haoa, Nao Jonaia, Randall M. China, Mariam Aghajana, Sarah Warburtonb, Jianing Wangc,
Raymond P. Wua, Fernando Gomezd, Joseph A. Looc,d, James A. Wohlschlegelc, Thomas M. Vondriskab, Jerry Pelletiere,
Harvey R. Herschmana,c, Jon Clardyf, Catherine F. Clarked, and Jing Huanga,1
aDepartment of Molecular and Medical Pharmacology and the Molecular Biology Institute, and Departments of bAnesthesiology, Medicine/Cardiology and
Physiology, and cBiological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; dDepartment of Chemistry and
Biochemistry, University of California, Los Angeles, CA 90095; eDepartment of Biochemistry, McGill University, Montreal, QC, Canada; and fDepartment of
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

Edited by Michael E. Phelps, University of California, Los Angeles, CA, and approved October 26, 2009 (received for review September 2, 2009)

Identifying the molecular targets for the beneficial or detrimental proteins protected from protease digestion through interacting
effects of small-molecule drugs is an important and currently unmet with their natural ligands such as DNA (9) and carbohydrates
challenge. We have developed a method, drug affinity responsive (5). However, previous experiments used large (DNA) or high-
target stability (DARTS), which takes advantage of a reduction in the affinity nanomolar hydrophilic (maltose) ligands, and both cases
protease susceptibility of the target protein upon drug binding. involve major conformational change in the host protein (10, 11).
DARTS is universally applicable because it requires no modification of It remained unclear whether protease susceptibility of the target
the drug and is independent of the mechanism of drug action. We protein would be different in the absence of large conforma-
demonstrate use of DARTS to identify known small-molecule–protein tional changes, e.g., upon binding of small hydrophobic drugs.
interactions and to reveal the eukaryotic translation initiation ma- Another question is whether the strategy would be amenable to
chinery as a molecular target for the longevity-enhancing plant lower-affinity ligands, e.g., clinically used drugs, which encom-
natural product resveratrol. We envisage that DARTS will also be pass a wide range of binding affinities, and hits identified from
useful in global mapping of protein–metabolite interaction networks chemical genetic screens, which typically are in the micromolar
and in label-free screening of unlimited varieties of compounds for range.
development as molecular imaging agents. As a proof-of-principle, we examined the well-studied immu-
nophilin FKBP12, which is the target for the nanomolar immuno-
aging 兩 label-free 兩 proteomics 兩 small molecules suppressant drugs rapamycin and FK506 (12). Proteolysis of
FKBP12 by the protease subtilisin was clearly decreased by the

D evelopment of effective and safe therapies is the holy grail of

medicine. For small-molecule drugs, which comprise most of
today’s medicines, a key challenge remains the identification of the
presence of rapamycin or FK506 (Fig. 1B). This protection is
selective: Incubation with wortmannin, a drug that does not bind
FKBP12, did not prevent proteolysis (Fig. 1B), and the drugs had
molecular targets underlying drug therapeutic effects and/or ad- no effect on subtilisin activity (Fig. S1 A). Because X-ray cocrystal
verse side effects. For small molecules discovered in phenotypic structures showed that binding of FK506 or rapamycin does not
screens, which are increasingly popular in chemical genetics studies, cause a conformational change in FKBP12 (12), our results above
identifying the biological (and potential therapeutic) targets, along suggest that drug binding alone is likely sufficient to stabilize the
with the off-targets, is a largely ad hoc affair; a systematic, widely bound protein in the protease-resistant state. This result could be
applicable and robust approach is badly needed (1, 2). Current due to a direct change in the protein folding–unfolding equilibrium
affinity-based target identification techniques are limited by the upon ligand binding (5).
necessity to modify each drug individually (without losing bioac- Given that rapamycin and FK506 are among the most potent
tivity), whereas indirect, non-affinity-based approaches depend on and specific drugs available, we decided to test whether DARTS
the drug’s ability to induce specific biochemical or cellular readouts would work similarly with a much weaker inhibitor. E4 is a
(3, 4) (supporting information (SI) Text). mid-micromolar kinase inhibitor of mTOR identified from a phe-
To overcome these limitations, we sought to develop a simple, notype-based chemical genetic screen. Indeed, proteolysis of
universally applicable target identification approach that analyzes mTOR by thermolysin was decreased by E4 in a dose-dependent
direct drug binding to targets. Given that a protein might become manner (Fig. 1C and Fig. S1B).
less susceptible to proteolysis when it is drug-bound than when it is
drug-free (5–7), we hypothesized that this phenomenon could be DARTS Using Complex Protein Mixtures. The experiments above
exploited for target identification. This would allow the protein established that DARTS can efficiently test, screen, or verify
target of a drug to be revealed, without requiring modification or drug–protein interactions when the protein is available in relatively
immobilization of the small molecule. Because our method, termed pure form. For DARTS to be generally useful as a discovery tool,
DARTS (drug affinity responsive target stability), is not limited by however, applicability to complex protein mixtures (such as cell
synthetic chemistry and is independent of any biological effects of
the drug (save its binding to the target protein), it can potentially
Author contributions: B.L., R.H., N.J., R.M.C., M.A., H.R.H., and J.H. designed research; B.L.,
be used to identify the target for any small molecule. R.H., N.J., R.M.C., M.A., J.W, and R.P.W. performed research; S.W., F.G., J.A.L., J.A.W.,
T.M.V., J.P., J.C., and C.F.C. contributed new reagents/analytic tools; B.L., N.J., R.M.C., M.A.,
Results H.R.H., and J.H. analyzed data; and B.L., R.H., N.J., R.M.C., M.A., J.A.L., T.M.V., H.R.H., J.C.,
DARTS Strategy and Proof-of-Concept. The basic strategy of DARTS and J.H. wrote the paper.

is shown in Fig. 1A. Binding of drugs is proposed to stabilize The authors declare no conflict of interest.
target proteins, either globally or locally, e.g., in a specific This article is a PNAS Direct Submission.
conformation or by simply masking protease recognition sites, Freely available online through the PNAS open access option.
thereby reducing protease sensitivity of the target protein. This 1To whom correspondence should be addressed. E-mail: [email protected].
idea is analogous to several familiar concepts, from DNase This article contains supporting information online at www.pnas.org/cgi/content/full/
resistance of DNA sites bound by transcription factors (8) to 0910040106/DCSupplemental.

21984 –21989 兩 PNAS 兩 December 22, 2009 兩 vol. 106 兩 no. 51 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0910040106
Fig. 1. The DARTS method for drug target identification. (A) Scheme of DARTS. (B) Proof of principle. Recombinant human FKBP12 was incubated with indicated
drugs and digested with subtilisin. (C) DARTS with a micromolar mTOR kinase inhibitor (E4). Purple arrow, recombinant human TOR fragments protected from
thermolysin proteolysis; *, nonspecific band.

lysates) would be desirable. To demonstrate feasibility, we per- Identification of a Molecular Target for Resveratrol Using DARTS.
formed DARTS using human Jurkat cells treated with didemnin B Next, we applied DARTS to identify a molecular target of resvera-
(DB), an anticancer marine natural product whose binding to trol, a compound in red grapes and wine known for various health
EF-1␣ had previously been well characterized (13). Given that benefits including lifespan extension (14). Although resveratrol
EF-1␣ is a highly abundant protein, we first tested whether in the influences the activities of many proteins, no direct molecular target
DARTS protocol DB would protect EF-1␣ from proteolysis and has been demonstrated. Low specific binding affinity as suspected
result in a detectable difference. Indeed, DARTS revealed a strong from its modest size and structure (Fig. 3A), poor potency, and
protected band at ⬇50 kDa in the proteolysed extracts of DB- potential requirement of the polyphenol groups for its activity have
treated cells (Fig. 2A), whereas no detectable difference was discouraged generation of affinity reagents for target identification.
observed in the same samples that underwent mock digestion. Also, even at saturating concentrations, resveratrol inhibits yeast
Examination of the protected band and the matching gel region growth only very weakly if at all (SI Text), making it a poor
of the control lane by mass spectrometry confirmed that EF-1␣ was candidate for target identification using fitness profiling strategies.
the primary protein present at higher abundance in the DB-treated DARTS with resveratrol-dosed yeast cell lysates revealed two
sample (Fig. 2B). This analysis does not exclude the possibility of silver-stained bands between the 15- and 20-kDa MW markers that
other protected targets of lower abundance that were not evident were more intense in the resveratrol-treated lysate postproteolysis
by eye on the gel. DB-concentration-dependent proteolytic protec- compared with vehicle control (Fig. 3B). Mass spectrometry anal-
tion of EF-1␣ was also observed by immunoblotting, both when ysis of both bands showed that eIF4A, along with several ribosomal
intact cells were treated with DB (Fig. 2C) and when the lysates of proteins, were enriched in the resveratrol-treated sample (Table S1
untreated cells were incubated with DB in vitro (Fig. S2). The and Dataset S1). This enrichment was confirmed by Western blot
generality of this approach is further supported by experiments analysis using the TAP-tagged (15) eIF4A yeast strain (Fig. S5).
using diverse protein–drug pairs ranging from nano- to micromolar: This finding suggests that resveratrol might directly bind to one or
mTOR-rapamycin, COX-2–celecoxib, and SCF E3 ubiquitin more proteins comprising the protein translation machinery. Po-
ligase-inhibitor (Fig. S3). Furthermore, DARTS is not enzyme tential direct binding was further supported by a target mutation
specific, and much higher overall digestion efficiency can be analysis, where a Tif1 A64Q point mutant confers resistance to
achieved by using other proteases while retaining protection of the resveratrol (Fig. 3C). Although the alanine is conserved throughout

PHARMACOLOGY
target protein (SI Text and Fig. S4). fungi and animals, plants have a glutamine at this position, and the

Fig. 2. DARTS using whole-cell lysate. (A) Intact Jurkat cells were treated with DB (1 ␮g/mL), and lysates were subjected to thermolysin digestion and Coomassie
(SimplyBlue)-staining. (B) Enrichment of EF-1␣ isoforms in the protected band from A revealed by mass spectrometry analysis (SI Text and Fig. S6). Red, protein
enriched ⬎2-fold with P value ⬍0.001; green, protein depleted ⬎2-fold with P value ⬍0.001; blue, unchanged protein. (C) DARTS detection via immunoblotting.
GAPDH was resistant to thermolysin under the condition and served as a loading indicator.

Lomenick et al. PNAS 兩 December 22, 2009 兩 vol. 106 兩 no. 51 兩 21985
Fig. 3. DARTS identifies a molecular target of resveratrol. (A) Chemical structure of resveratrol. (B) Yeast cell lysates and human HeLa cell lysates were each treated
with resveratrol in vitro, followed by thermolysin digestion and silver staining. Protected bands of similar size were detected. (C) Resveratrol protects the wild-type
eIF4A, but not the A64Q-substituted eIF4A mutant protein, from proteolysis. (D) Resveratrol inhibits eIF4A-dependent translation in HEK 293 cells as assayed by
bicistronic translation reporters. The EMCV IRES requires the eIF4A and eIF4G subunits of eIF4F, whereas the HCV IRES does not (55). (E) eIF4A is required for longevity
in resveratrol-treated animals. Resveratrol (50 ␮M) lengthens the lifespan of wild-type N2 worms fed control (gfp) RNAi (green), but not worms fed eIF4A (inf-1) RNAi
(red) or daf-16 RNAi (blue). gfp(RNAi), mVeh ⫽ 19 (n ⫽ 74), mRSV ⫽ 20 (n ⫽ 78), ***, P ⫽ 0.0006; inf-1(RNAi), mVeh ⫽ 26 (n ⫽ 76), mRSV ⫽ 24 (n ⫽ 79), P ⫽ 0.4687; daf-16(RNAi),
mVeh ⫽ 17 (n ⫽ 78), mRSV ⫽ 17 (n ⫽ 76), P ⫽ 0.3305. m, mean lifespan (days of adulthood); n, number of animals tested.

bulkier side chain is hypothesized to protect plant eIF4A from found that cap-dependent translation and EMCV IRES-mediated
resveratrol inhibition by minimizing self binding. translation, both of which require eIF4A, were inhibited in a
The molecular mechanisms underlying resveratrol’s lifespan ef- dose-dependent manner by resveratrol, whereas translation from
fect have been controversial (16, 17), and whether Sir2 serves as a the eIF4A-independent, HCV IRES was unaffected (Fig. 3D).
direct target for resveratrol is an interesting problem that is being These results indicate that resveratrol specifically inhibits eIF4A- or
pursued. On the other hand, it is interesting to note that multiple eIF4G-dependent translation initiation and does not impinge on
genome-wide studies in Saccharomyces cerevisiae and Caenorhab- other translation initiation factors or on translation elongation.
ditis elegans have found knockouts or knockdowns of eIF4A and Finally, we asked whether eIF4A is required for resveratrol’s
several ribosomal proteins to have significant increases in lifespan longevity effect. Whereas resveratrol lengthens the lifespan of
(18). Our finding of resveratrol-mediated protection of eIF4A and wild-type worms (Fig. 3E), as reported previously (14), this lon-
ribosomal proteins by DARTS suggested that the protein transla- gevity effect is lost in eIF4A knockdown worms (Fig. 3E), consistent
tion machinery may be a molecular target of resveratrol in lifespan with eIF4A being a physiological target of resveratrol. Interestingly,
extension. To test this notion, we first asked whether resveratrol has the longevity effect of resveratrol appears to require daf-16 (Fig.
a specific effect on protein translation. Using bicistronic dual- 3E), the Forkhead transcription factor that mediates lifespan ex-
luciferase reporters to monitor cap-dependent translation (which tension by the insulin/IGF-1 pathway (19), reminiscent of its
requires initiation factors) and translation mediated by IRESs requirement for longevity in eIF4G knockdown animals (20).
(which exhibit differing requirements for initiation factors), we Taken together, it is plausible that resveratrol increases lifespan by

21986 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0910040106 Lomenick et al.

Fig. 4. DARTS using cDNAs. (A) Plasmid cDNA is used to program IVT for DARTS. (B) FKBP12-rapamycin protects translated mTOR fragment in DARTS.
Streptavidin-HRP was used to detect biotin-Lys incorporated into the translation product. ␤-Actin was less susceptible to thermolysin under the condition and
served as loading indicator. (C) DARTS with IVT FLAG-tagged mTOR.

direct inhibition of translation initiation, through binding to eIF4A Discussion

and/or one or more ribosomal proteins in the preinitiation complex. Developing new methods for drug target identification is an area of
However, this interpretation should be taken with an important intense interest, and both experimental and computational ap-
caveat because the eIF4A-knockdown worms show a significantly proaches have been developed (28, 29). Previous methods for drug
enhanced lifespan (beyond the extension produced by resveratrol in target identification have had substantial success, but many limi-
wild-type worms). Furthermore, it is possible that knocking down tations remain. Traditionally, affinity chromatography has played a
an initiation factor like eIF4A will affect the expression level of major role in the identification of the binding targets for many
many other proteins that could be targets. Our findings also point biologically active small molecules and natural products (SI Text).
to eIF4A (and possibly other translation factors) as a previously In addition to affinity chromatography, many new methods for drug
uncharacterized druggable target for antiaging therapy. Several target identification have been developed, ranging from biochem-
potent eIF4A inhibitors have recently been identified (21), and it istry to genetics, proteomics, and imaging (30–36) (SI Text). All
will be interesting to test them for potential longevity effects. current target identification methods are of two main categories:
affinity-based methods, which detect the direct binding of the drug
DARTS Using Proteins Generated from cDNA. The utility of DARTS to its target(s); and phenotype-based methods, which infer drug
in complex mixtures suggests that potential drug targets can be targets/pathways from the physiological responses or biochemical
identified by using a wide range of biological systems, and the signatures the drugs produce.
method is unlimited by the availability and coverage of knockout
(or knockdown) libraries and genome arrays for model organ- Affinity-Based Target Identification Methods. Affinity-based meth-
isms. The limiting aspect of DARTS analysis is likely to be ods include matrix-based affinity detection and matrix-free affinity
sensitivity of detection by mass spectrometry or other potential labeling. Matrix-based affinity detection fuses the small molecule of
methods (as in affinity chromatography). This limitation is being interest to a solid support or capturable moiety such as biotin (SI
increasingly alleviated with the development of more sensitive Text). Such matrix-based methods must satisfy three basic condi-
analytical tools. Nonetheless, we tested whether DARTS can be tions: (i) that the small molecule contains a derivatizable function-
applied to a complementary unbiased platform, namely using ality, (ii) that bioactivity/binding specificity of the small molecule is
proteins generated from cDNAs by in vitro transcription/ unaffected by the derivatization, and (iii) that the matrix does not
translation (IVT) (Fig. 4A). hinder the binding of target protein to drug. The latter two criteria
cannot be predicted a priori. Matrix-free affinity labeling relies on

PHARMACOLOGY
Reticulocyte lysate IVT is a powerful technique routinely used
the incorporation of radioisotope, photoreactive, or fluorescent
in studies of protein function and is readily adaptable to express
labels into the small molecule of interest (SI Text) and must also
proteins in a high-throughput manner (22). As a test case, we used
satisfy criteria one and two above. In both affinity chromatography
the human mTOR (mammalian target of rapamycin) protein, which
and matrix-free methods, proteins are incubated with the modified
is an important target (23, 24) and had been identified on the basis small molecule, and the binding proteins are revealed by mass
of its association with the FKBP12-rapamycin complex (see refer- spectrometry after gel electrophoresis. Genetic and other versions
ences in ref. 25). As shown in Fig. 4B, an IVT mTOR fragment of matrix-based affinity chromatography, e.g., yeast three-hybrid
containing the FKBP-rapamycin-binding domain (25) was pro- (37) and phage display cloning (38), require tagged small molecules
tected from thermolysin digestion by the presence of FKBP12- as well. Thus, all current affinity methods are limited to small
rapamycin, whereas the S2035T-substituted mTOR that abolishes molecules that contain derivatizable functionalities and whose
rapamycin binding (25) was not protected. IVT TOR proteins were bioactivity/binding is unaffected by the modification (SI Text).
labeled with biotin-Lys in this experiment, but other forms of amino Because DARTS does not require labeled ligands and instead uses
acids could also be used. Alternatively, IVT proteins could be ‘‘native’’ (i.e., unmodified) small molecules for binding, it is not
detected through an epitope tag without incorporation of any limited by chemistry and can potentially be used for any small
artificially labeled amino acids. For example, FLAG-tagged full- molecule.
length mTOR protein was also a robust source of protein for the
DARTS method (Fig. 4C). These results further demonstrate the Affinity-Free Target Identification Methods. Indirect, non-affinity-
versatility of DARTS and suggest nearly unlimited possibilities for based approaches, which infer drug targets/pathways from the
screening cDNA libraries (26) and genome-wide collections of physiological responses or biochemical signatures the drugs pro-
epitope-fused proteins (15, 27) using DARTS to systematically duce, have also been developed. For example, classical genetics
analyze small-molecule–protein interactions. relies on the isolation of drug-resistant mutations (39) or gene

Lomenick et al. PNAS 兩 December 22, 2009 兩 vol. 106 兩 no. 51 兩 21987
dosage effects (40), and several genome-wide methods also rely on Effect of in Vivo Protein Stability on DARTS. We rely on in vitro
fitness changes (3, 41–44). An inherent limitation of these methods proteolysis using exogenous proteases in our DARTS method.
is that they are applicable only to drugs that affect cell growth/ Protein stability in vivo on the other hand is a much more
viability. Another powerful approach, genome-wide expression complicated problem. Because degradation of proteins inside the
profiling (4, 45), on the other hand, is applicable only to drugs that cell is predominantly carried out by supramolecular machines,
induce major transcriptome changes. These genetic and large-scale known as the proteasomes and aggresomes, and is elaborately
‘‘omic’’ profiling approaches are also primarily limited to yeast or controlled by posttranslational modifications such as phosphoryla-
other simple, well-characterized model organisms. Moreover, the tion and ubiquitinylation, protein stability in vivo is largely unpre-
‘‘readout’’ is often far downstream from the drug target. dictable. Indeed, in vivo stability of proteins upon drug/ligand
binding is highly idiosyncratic in the literature; drug binding has
Advantages of DARTS. Like affinity chromatography, DARTS relies been shown to both increase and decrease proteolytic susceptibility
on the affinity between a drug molecule and its protein target and of the target protein (6, 47, 51, 52). For instance, whereas unstable
thereby is able to pinpoint direct binding partner(s) of the drug. The FRB domain and FKBP12 mutants are stabilized by the presence
key advantage of DARTS, however, is that because it does not of ligands (6, 51) and topoisomerase-1 is destabilized by campto-
require labeled ligands and instead uses ‘‘native’’ (i.e., unmodified) thecin (47), binding of estrogen receptor ligands each affects
small molecules for binding, it is not limited by chemistry and can receptor stability differently (53). In any event, this information
potentially be used to identify binding targets for any small mole- would be useful in conjunction with DARTS for elucidating the
cule. Additionally, unlike cell-based methods, DARTS is com- molecular mechanisms of action of drugs.
pletely independent of any effects of the drug on the system, and is
therefore compatible with any mechanism of action, making it Additional Applications of DARTS. Beyond drugs, we envisage that
useful for any small molecule of interest. Moreover, DARTS can be DARTS will also be useful for global mapping of protein–
performed by using any cell or tissue type from any organism and metabolite interaction networks and in elucidating potential pro-
is thus not limited by the availability and coverage of knockout (or tein targets for small molecules found in food or dietary supple-
knockdown) libraries and genome arrays for model organisms. ments. DARTS may also be useful in identifying a wide range of
Once identified, potential drug targets can be confirmed by small molecules that can be developed into a new genre of molec-
functional studies, and kinetics and affinities of the interactions can ular imaging agents. Pharmaceutical agents almost always interact
be measured by using a variety of analytical methods. Although with the active sites of enzymes or the ligand-binding sites of
biophysical methods (i.e., surface plasmon resonance, isothermal receptors. The design of most small-molecule molecular imaging
titration calorimetry, etc.) are traditionally used to analyze direct probes usually begins with modification of the structure of known
binding, DARTS proves to be a fast and robust method to deter- drugs. However, enzyme active sites and ligand-binding sites rep-
mine direct binding of a small molecule (or metabolite) without resent only a very small percentage of the tertiary structures of
requiring large amounts of pure protein and is even amenable to target proteins. Small molecules that bind tightly and specifically to
using whole-cell lysates. sites other than the active site or the ligand-binding site on a protein
and are detectable by DARTS would provide initial ‘‘hits’’ from
Potential Limitations of the DARTS Method. First, the binding affinity which probes that can stoichiometrically measure protein concen-
of the drug to its target may be a limiting factor. To date, our trations by fluorescence, positron emission tomography, single-
experiments suggest that DARTS is effective for molecules with photon emission tomography, and other molecular imaging tech-
inhibitory concentrations across many orders of magnitude, up to nologies could be developed through conventional medicinal
high-micromolar. Second, a potential fundamental limitation for chemistry or secondary chemical library procedures.
DARTS is that a protein’s susceptibility to proteolysis is determined
by its conformational energy landscape, and it has been demon- Materials and Methods
strated that a small number of evolutionarily selected proteins (e.g., Reagents and Plasmid Constructs. See SI Text for additional information.
stress proteins) are quite refractory to protease digestion (46).
Third, drug binding may change the protease susceptibility of DARTS with Pure Proteins. See SI Text for additional information.
nontarget proteins, such as those that interact with or are part of
complexes containing the target. But this result could be an DARTS with Complex Protein Mixtures. For Fig. 2 A and C, intact Jurkat cells were
advantage of the DARTS approach as well, insofar as it would treated with DB from 100 pg/mL to 1 ␮g/mL or DMSO control for 30 min. Cells
provide information about protein complexes that are dissociated were lysed (without washing, in these experiments) with M-PER (Pierce) supple-
(or formed) upon drug binding. Drug binding in vivo might also mented with protease and phosphatase inhibitors. After centrifugation (14,000
rpm using Beckman Coulter Microfuge 22R with F241.5P rotor, 15 min), lysates
increase proteolytic susceptibility of the target protein (47) (see SI
were diluted to the same final volume and protein concentration with M-PER and
Text). This would—in the DARTS protocol—also identify target proteolysed in reaction buffer [50 mM Tris䡠HCl (pH 8.0), 50 mM NaCl, 10 mM
proteins of the small molecule being analyzed. A small-molecule CaCl2]. All steps were performed on ice or at 4 °C to help prevent premature
effector that destabilizes a protein could, of course, also be iden- protein degradation. Each sample was then quickly warmed to room tempera-
tified by DARTS. ture and proteolysed with 1 ␮g of thermolysin for every 15 ␮g of lysate for 10 min.
An extrinsic limiting aspect of DARTS analysis is likely to be To stop proteolysis, 0.5 M EDTA (pH 8.0) was added to each sample at a 1:10 ratio,
sensitivity of detection by mass spectrometry (as in affinity chro- mixed well, and placed on ice.
matography). Although DB-mediated protection of EF-1␣ was For DARTS using yeast cell lysates incubated in vitro with resveratrol (Fig.
visualized by eye on a stained gel in Fig. 2 A, this will not necessarily 3B), S. cerevisiae BY4742 cells were used (see SI Text and Fig. S7).
be the case with many target proteins of lower abundance. Quan-
titative imaging or densitometry could prove useful with DARTS Mass Spectrometry Analysis. Gel bands were cut out and prepared for mass
spectrometry analysis with trypsin digestion as described in SI Text. Peptides
to assist in finding more subtle differences in protein abundance.
were analyzed by LC/MS/MS on a Thermo LTQ-Orbitrap mass spectrometer
Furthermore, proteomic techniques including 2D gels (48), DIGE with an Eksigent LC pump. For quantitative comparison of protein and pep-
(49), and gel-free approaches like MudPIT (50) would likely tide abundances, MS spectra were analyzed by using the differential work-
provide even greater sensitivity in conjunction with DARTS. flow of Rosetta Elucidator (Rosetta Inpharmatics) (54). Annotation was per-
Finally, the use of cDNA libraries to express proteins in cell culture formed using PeptideTeller and ProteinTeller (see SI Text).
or by IVT, as demonstrated in Fig. 4, also provides viable alterna-
tives. In Vivo Translation Assays. See SI Text for additional information.

21988 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0910040106 Lomenick et al.

Lifespan Analysis. C. elegans lifespan analysis was conducted with N2 worms synthesized by IVT using 0.5 ␮g of pcDNA3.1-FLAG-hTOR vector in a 50-␮L
at 20 °C (see SI Text). reaction at 30 °C for 3 h (Promega TnT T7 Quick Coupled Transcription/
Translation System). DARTS was performed by using 6 ␮L of translated
DARTS Using Proteins Generated by Rabbit Reticulocyte Lysate In Vitro Tran- lysate in a 10-␮L total reaction volume in reaction buffer [50 mM Tris䡠HCl
scription/Translation (IVT) System. For Fig. 4B, IVT was performed by using (pH 8.0), 50 mM NaCl, 10 mM CaCl2] with 1 ␮M rapamycin or 1 ␮M FK506
Promega TnT T7 Quick Coupled Transcription/Translation System, with 0.5 or cotreatment with 50 nM FKBP12 (preincubated on ice for 30 min to allow
␮g of pcDNA3.1-hTOR1968C (encoding human mTOR amino acid 1968 to complex formation) and incubated on ice for 45 min. Proteolysis was
C-ter) or pcDNA3.1-hTOR1968C S2035T [encoding corresponding rapamy- performed with 1 ng of thermolysin at room temperature for 40 min and
cin-resistant mutant (25)] vectors (see SI Text), and 2 ␮L of ␧-biotin-Lys- stopped with 1 ␮L of 0.5 M EDTA (pH 8.0).
tRNA (Transcend tRNA; Promega) in 50-␮L reaction at 30 °C for 3 h. DARTS
was performed by using 5 ␮L of translated lysate in a 10-␮L total reaction
ACKNOWLEDGMENTS. We thank Ken Houk and Ray Deshaies for comments
volume in 50 mM Tris䡠HCl (pH 8.0), 50 mM NaCl, 10 mM CaCl2, with 1 ␮M
and discussions and David Sinclair and Matt Kaeberlein for advice on
rapamycin, 50 nM FKBP12, or 50 nM FKBP12 ⫹ 1 ␮M rapamycin (preincu- lifespan assays. This work was partially supported by National Institutes of
bated on ice for 30 min to allow complex formation) and incubated on ice Health National Cancer Institute Grant R01 CA124974 and by American
for 30 min. Proteolysis was performed with 2 ng of thermolysin at room Cancer Society Grant RSG-07-035-01-CCG. B.L. and M.A. were trainees of
temperature for 20 min, and stopped with 1 ␮L of 0.5 M EDTA (pH 8.0). the National Institutes of Health UCLA Chemistry–Biology Interface Pre-
For Fig. 4C, N-terminal FLAG-tagged full-length human TOR protein was doctoral Training Program T32 GM008496.

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