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Protein analysis and proteomics(Part 2 of 2)
Many of the images in this powerpoint presentationare from Bioinformatics and Functional Genomicsby Jonathan Pevsner (ISBN 0-471-21004-8). Copyright © 2003 by John Wiley & Sons, Inc.
These images and materials may not be usedwithout permission from the publisher. We welcomeinstructors to use these powerpoints for educationalpurposes, but please acknowledge the source.
The book has a homepage at http://www.bioinfbook.orgIncluding hyperlinks to the book chapters.
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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 celland/or all the proteins in an organism
Large-scale protein analysis2D protein gelsYeast two-hybridRosetta Stone approachPathways
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Classical biochemical approach
Identify an activityDevelop a bioassayPerform a biochemical purification
Strategies: size, charge, hydrophobicityPurify protein to homogeneityClone cDNA, express recombinant protein
Grow crystals, solve structure (Wednesday)
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Two-dimensional protein gels
First dimension: isoelectric focusing
Second dimension: SDS-PAGE
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Two-dimensional protein gels
First dimension: isoelectric focusing
Electrophorese ampholytes to establisha 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)
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Two-dimensional protein gels
Second dimension: SDS-PAGE
Electrophorese proteins through an acrylamidematrix
Proteins are charged and migrate through an electric field v = Eq / d6r
Conditions are denaturing
Can resolve hundreds to thousands of proteins
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Proteins identified on 2D gels (IEF/SDS-PAGE)
Direct protein microsequencing byEdman 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
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Evaluation of 2D gels (IEF/SDS-PAGE)
Advantages:Visualize hundreds to thousands of proteinsImproved identification of protein spots
Disadvantages:Limited number of samples can be processedMostly abundant proteins visualizedTechnically difficult
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Affinity chromatography/mass spec
Bait proteinGST
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Affinity chromatography/mass spec
Bait proteinGST
Add yeast extractProtein complexes bindMost proteins do not bind
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Affinity chromatography/mass spec
Bait proteinGST
EluteRun gelMALDI-TOFIdentify complexes
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Affinity chromatography/mass spec
Data on complexes deposited in databases
http://yeast.cellzome.comhttp://www.bind.ca
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Evaluation of affinity chromatography/mass spec
Advantages:Thousands of protein complexes identifiedFunctions can be assigned to proteins
Disadvantages:False negative resultsFalse positive results
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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
Bait proteinGST
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Affinity chromatography/mass spec
False positives:• sticky proteins
Bait proteinGST
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The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
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The yeast two-hybrid system
Reporter gene
Prey proteinDNA activation
Prey proteinDNA activation
Prey proteinDNA activation Prey protein
DNA activation
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The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
Prey proteinDNA activation
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The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
Prey proteinDNA activation
Isolate and sequence the cDNAof the binding partner you have found
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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 identifiedFunctions can be assigned to proteins
Disadvantages:Detects only pairwise protein interactionsFalse-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
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The Rosetta Stone approach
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Marcotte et al. (1999) and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes, but occasionally theyare fused into a single gene.
By scanning many genomes for examples of “fusedgenes,” several thousand protein-protein predictionshave been made.
Yeast topoisomerase II
E. coligyrase B
E. coligyrase A
The Rosetta Stone approach
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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
ExPASyProNetEcoCyc: E. coli pathwaysMetaCyc: 450 pathways, 158 organismsKEGG: Kyoto Encyclopedia of Genes & Genomes
Issues:Is the extrapolation between species valid?Have orthologs been identified accurately?False positive, false negative findings
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