Transcript 91.510_ch8_part2

Protein analysis and proteomics
(Part 2 of 2)
Copyright notice
Many of the images in this powerpoint presentation
are from Bioinformatics and Functional Genomics
by Jonathan Pevsner (ISBN 0-471-21004-8).
Copyright © 2003 by John Wiley & Sons, Inc.
These images and materials may not be used
without permission from the publisher. We welcome
instructors to use these powerpoints for educational
purposes, but please acknowledge the source.
The book has a homepage at http://www.bioinfbook.org
Including hyperlinks to the book chapters.
Proteomics: High throughput protein analysis
Proteomics is the study of the entire collection
of proteins encoded by a genome
“Proteomics” refers to all the proteins in a cell
and/or all the proteins in an organism
Large-scale protein analysis
2D protein gels
Yeast two-hybrid
Rosetta Stone approach
Pathways
Page 247
Classical biochemical approach
Identify an activity
Develop a bioassay
Perform a biochemical purification
Strategies: size, charge, hydrophobicity
Purify protein to homogeneity
Clone cDNA, express recombinant protein
Grow crystals, solve structure (Wednesday)
Page 247
Two-dimensional protein gels
First dimension: isoelectric focusing
Second dimension: SDS-PAGE
Page 248
Page 249
Two-dimensional protein gels
First dimension: isoelectric focusing
Electrophorese ampholytes to establish
a pH gradient
Can use a pre-made strip
Proteins migrate to their isoelectric point
(pI) then stop (net charge is zero)
Range of pI typically 4-9 (5-8 most common)
Page 248
Two-dimensional protein gels
Second dimension: SDS-PAGE
Electrophorese proteins through an acrylamide
matrix
Proteins are charged and migrate through an
electric field
v = Eq / d6prh
Conditions are denaturing
Can resolve hundreds to thousands of proteins
Page 248
Page 249
Proteins identified on 2D gels (IEF/SDS-PAGE)
Direct protein microsequencing by
Edman degradations
-- done at Hopkins, other cores
-- typically need 5 picomoles
-- often get 10 to 20 amino acids sequenced
Protein mass analysis by MALDI-TOF
-- done at core facilities
-- often detect posttranslational
modifications
-- matrix assisted laser desorption/ionization
time-of-flight spectroscopy
Page 250-1
Page 252
Page 253
Page 253
Page 254
Evaluation of 2D gels (IEF/SDS-PAGE)
Advantages:
Visualize hundreds to thousands of proteins
Improved identification of protein spots
Disadvantages:
Limited number of samples can be processed
Mostly abundant proteins visualized
Technically difficult
Page 251
Affinity chromatography/mass spec
GST Bait protein
Page 252
Affinity chromatography/mass spec
GST Bait protein
Add yeast extract
Protein complexes bind
Most proteins do not bind
Page 252
Affinity chromatography/mass spec
GST Bait protein
Elute
Run gel
MALDI-TOF
Identify complexes
Page 252
Affinity chromatography/mass spec
Data on complexes deposited in databases
http://yeast.cellzome.com
http://www.bind.ca
Page 252
Page 252
Page 252
Evaluation of affinity chromatography/mass spec
Advantages:
Thousands of protein complexes identified
Functions can be assigned to proteins
Disadvantages:
False negative results
False positive results
Page 253-254
Affinity chromatography/mass spec
False negatives:
• Bait must be properly localized and
in its native condition
• Affinity tag may interfere with function
• Transient protein interactions may be missed
• Highly specific physiological conditions
may be required
• Bias against hydrophobic, and small proteins
GST Bait protein
Page 253
Affinity chromatography/mass spec
False positives:
• sticky proteins
GST Bait protein
Page 253
The yeast two-hybrid system
Bait protein
DNA Binding
Reporter gene
Page 255
The yeast two-hybrid system
Prey protein
DNA activation
Prey protein
DNA activation
Reporter gene
Prey protein
DNA activation
Prey protein
DNA activation
Page 255
The yeast two-hybrid system
Bait protein
DNA Binding
Prey protein
DNA activation
Reporter gene
Page 255
The yeast two-hybrid system
Bait protein
DNA Binding
Prey protein
DNA activation
Reporter gene
Isolate and sequence the cDNA
of the binding partner you have found
Page 255
http://depts.washington.edu/sfields/yplm/data/index.html
red = cellular role & subcellular localization of interacting proteins are identical;
blue = localizations are identical; green = cellular roles are identical
Evaluation of the yeast two-hybrid system
Advantages:
Thousands of protein complexes identified
Functions can be assigned to proteins
Disadvantages:
Detects only pairwise protein interactions
False-negative results (as for affinity chromatography)
-- bait may be mislocalized
-- transient interactions may be missed
-- some complexes require special conditions
-- bias against hydrophobic proteins
False-positive results
-- some proteins may be sticky
-- bait protein may auto-activate a reporter
Page 256
The Rosetta Stone approach
Marcotte et al. (1999) and other groups hypothesized
that some pairs of interacting proteins are encoded by
two genes in many genomes, but occasionally they
are fused into a single gene.
By scanning many genomes for examples of “fused
genes,” several thousand protein-protein predictions
have been made.
Page 258
The Rosetta Stone approach
Yeast topoisomerase II
E. coli
gyrase B
E. coli
gyrase A
Page 258
http://depts.washington.edu/sfields/yp_project/index.html
6,217 yeast proteins
Experimental data (500 links)
Related metabolic function (2,000 links)
Related phylogenetic profiles (20,000 links)
Rosetta Stone method (45,000 links)
Correlated mRNA expression (26,000 links)
Marcotte et al. (1999) Nature 402:83
Pathway maps
A pathway is a linked set of biochemical reactions
ExPASy
ProNet
EcoCyc: E. coli pathways
MetaCyc: 450 pathways, 158 organisms
KEGG: Kyoto Encyclopedia of Genes & Genomes
Issues:
Is the extrapolation between species valid?
Have orthologs been identified accurately?
False positive, false negative findings
Page 258
Page 257
Page 257
Page 260
Page 262
Page 262