Last Lecture Slides
Last Lecture Slides
Last Lecture Slides
Carbohydrates
Supplemental Slides
Biochem 5613
Figures: Lehninger Principles of Biochemistry 6th Ed., Nelson & Cox
Essentials of Biochemistry, 3rd Ed. OSU Custom Edition
Voet & Voet, Biochemistry 3rd Ed.
Voet, Voet & Pratt, Fundamentals of Biochemistry 2nd Ed.
Berg, Tymoczko, & Stryer, Biochemistry 2001
Glycoproteins
N-linked glycoproteins: Large, heterogeneous
oligosaccharides linked to asparagine side chains
O-linked glycoproteins: Smaller carbohydrates (usually
1-5 saccharides) linked to hydroxyl groups of Ser and/or
Thr side chains
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Glycoproteins
Carbohydrate may be a large amount of the glycoprotein mass
Carbohydrate is
hydrophilic; usually on
surface of a folded protein
Glycoproteins
Oligosaccharide substituents of proteins are highly dynamic
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Glc3Man9GlcNac2
Man
Man
Man
GlcNAc
GlcNAc
Man
Man
Man
Man
Man
Glc
Glc
Glc
N-Linked Glycosylation
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O-Linked Glycosylation
O-linked glycosylation of proteins occurs in the Golgi
apparatus
Sugar groups form a glycosidic bond with the side chain
hydroxyl group of Ser or Thr
One major purpose is immunological identification:
For example: Mucins are heavily glycosylated
proteins which are a significant component of mucus
Many cell-surface proteins are O-glycosylated
O-Linked Glycosylation
Core is not as conserved as in N-linked glycosylation
Gal-GalNAc-Ser/Thr
is the most commonly
observed core
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Protein Sequencing
Chapter 3.4
Biochem 5613
Figures: Lehninger Principles of Biochemistry 6th Ed., Nelson & Cox
Essentials of Biochemistry, 3rd Ed. OSU Custom Edition
Voet & Voet, Biochemistry 3rd Ed.
Voet, Voet & Pratt, Fundamentals of Biochemistry 2nd Ed.
Berg, Tymoczko, & Stryer, Biochemistry 2001
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Complete conversion
Ser, Thr
H3N+
COOH
Dehydrate
H3N+
COOH
Dehydroalanine
Partial degradation of Cys, Ser, Thr, and complete degradation
of Trp; do NOT get accurate numbers for these amino acids!
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Insulin
First sequenced protein (Sanger)
Two chains complicates N-terminal
identification
Three disulfide bonds
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Protein Sequencing
Proteins are typically cleaved in multiple ways to allow the
generation of overlapping peptide fragments
Fragmentation of Proteins
Determining the sequence of very large proteins is complicated
It is common to break a protein into smaller peptide segments
to allow for easier sequencing
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Cyanogen Bromide
1)
3)
Methyl thiocyanate
leaving group
2)
Hydrolysis
4)
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Protease Terminology
Peptidase: breaks peptide bond
Exopeptidase: breaks peptide bond on a terminal amino acid
(exterior peptide bond)
Endopeptidase: breaks peptide bond somewhere in the
middle of (inside) the sequence
Carboxypeptidase:
Exopeptidase that hydrolyzes peptide bond at the C-terminus
(carboxyl terminus)
Aminopeptidase:
Exopeptidase that hydrolyzes peptide bond of the N-terminal
residue (amino terminus)
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Protease Specificities
Endo Lys-C
Endo Glu-C
Endo Asp-N
Name based on
specificity and
cleaved bond
Carboxypeptidase A
Trypsin
Aminopeptidase
Chymotrypsin
Cyanogen Bromide
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Peptide Sequencing
Edman Degradation
= Edmans reagent
Phenylisothiocyanate
Peptide Sequencing
Edman Degradation
Peptides can undergo multiple
cycles of Edman degradation to
identify entire sequence
Edman degradation can be
automated: protein sequencers
can carry out approximately one
round per hour
Up to 50 amino acids can be
sequenced fairly reliably before
small errors build up
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Ion source
Mass analyzer
Detector
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Observed =
m+z
z
HCCA
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H4
11237
H2B H2A
13496 13951
H3(pT118)
15354
m/Z
ESI vs MALDI
Electrospray MS: Syringe full of protein/peptide solution
Ionize by concentrating charges in vacuum
Highly charged spectrum
Often connected to liquid chromatography systems
MALDI MS: Crystallize protein/peptide in matrix
Ionize using laser
Tend to see the parent mass
Simpler analysis of mixtures of ions
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MALDI/Carboxypeptidase Sequencing
Time
series
Figure from John J. Lennon, ABRF News, 1997
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MS/MS Sequencing
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