2. Electrophoretic separation of proteins by charge (isoelectric

1
PROTEOMICS LARGE-SCALE PROTEIN IDENTIFICATION
ANALYSIS
2D-gel electrophoresis mass spectrometry
1. Isolate proteins from tissue (organism,
condition) of interest
2. Electrophoretic separation of proteins by
charge (isoelectric focusing) and by size
(SDS-PAGE)
3. Peptide fragmentation of individual protein
(with proteases eg trypsin)
Fig. 6.10
see Fig. 6.11
2
4. Determine precise peptide mass by MALDI-TOF
(matrix-assisted laser desorption ionization
time of flight) mass spectrometry
5. Compare aa sequences to genomic data to
correlate protein with its gene
Fig. 6.12
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STRUCTURAL PROTEOMICS
- large-scale determination of protein structures
Start with gene of interest cloning,
expression, purification of protein
X-ray crystallography
- crystallize protein X-ray diffraction analysis
NMR spectroscopy - for small proteins or
domains (in solution)
Protein data bank www.rcsb.org/pdb/
Nov 2000 13,750 structures
Nov 2001 16,550
Nov 2006 40,132
Nov 2008 54,559
Nov 2009 61,418
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Analysis of protein structures and sequences
eg. ExPASy Expert Protein Analysis System
www.expasy.org
(Swiss-Prot database at Swiss Institute of
Bioinformatics)
Identification of - protein motifs, catalytic
centres
- binding to ligands, drugs
- interaction with other macromolecules
- relatedness to other proteins (homology
modelling)
clues from protein sequence/structure about
biological function
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How to find proteins that interact with protein
of interest?
1. Phage display
- generate phage library producing collection of
fusion proteins between phage coat protein
test protein from genome of interest
- hybrid protein will be displayed on outer
surface of phage
- then screen library to find ones having
expressed protein which interacts with test
protein of interest
Fig. 6.14
6
2. Yeast 2-hybrid system
Background info about transcription in eukaryotes
Activator domain
Transcription factors have 2 domains
DNA binding domain
gene
mRNA
Regulatory cis-element
But if bait prey interact to bring TF
domains close together, then transcription occurs
If TF domains physically separated, no
transcription
mRNA
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Determining protein-protein interactions using
yeast 2-hybrid system
- use separate vectors to prepare 1 bait
fused to DNA binding domain of a yeast
transcription factor
2 shotgun library of possible prey fused to
activation domain of yeast TF
- fuse (1) to gene for protein X bait
prey generate library where (2) is fused to
random coding sequences from organism of interest
(eg. human)
Fig. 6.15
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- co-transform yeast cells (which lack this
transcription factor TF)
Fig. 6.15
if protein X and prey (from library) interact,
the 2 domains of yeast TF will be close together
( functional), so activate reporter gene
eg if use lacZ reporter gene blue colour of
yeast colony
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3. Affinity column chromatography
- protein B (bait) attached on column to fish
out the protein (or proteins) which specifically
bind to it
If bait protein does not interact directly with
protein(s) in a complex, they may not be isolated
Fig.6.18
or use co-immunoprecipitation
(p.182)
Fig.6.17
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3. Bioinformatics approach to predict
protein-protein interactions
- search for one large gene in organism X vs. two
separate smaller genes in Y
his2
his10
in E.coli
in yeast
HIS2
Fig.6.19
- based on premise that composite
(naturally-fused) proteins have direct physical
interaction (or functional association eg. role
in same biochemical pathway)
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Fusion detection algorithms used to search
complete genomes of E.coli, Haemophilus,
Methanococcus yeast
- found 215 cases of fusion vs. split state
- lines connect 2short genes in E.coli which
have 1 long gene as counterpart in Haemophilus,
Methanococcus or yeast
- such genes which are close together (eg. same
operon) are shown by small circles
Nature 402 86, 1999
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Yeast protein-protein interaction map (from
experimental data)
- lines connecting dots represent known protein-
protein interactions
2002
colour-coded for biological function
2001
Fig. 6.20
red dots essential proteins (so knockout is
lethal)
green non-lethal orange slow growth yellow
unknown effect
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Protein interaction network in Huntingtons
disease
- triplet repeat (CAG) expansion disease (p.510)
Max-Delbruck Centrum Berlin
14
Marcotte Nature 40283, 1999