Protein analysis and proteomics (Part 2 of 2)

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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 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

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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 / d6  r  Conditions are denaturing Can resolve hundreds to thousands of proteins Page 248

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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

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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 Bait protein GST Page 252

Affinity chromatography/mass spec Bait protein GST Add yeast extract Protein complexes bind Most proteins do not bind Page 252

Affinity chromatography/mass spec Bait protein GST Elute Run gel MALDI-TOF Identify complexes Page 252

Affinity chromatography/mass spec Data on complexes deposited in databases 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

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 protein GST Page 253

Affinity chromatography/mass spec False positives: sticky proteins Bait protein GST Page 253

The yeast two-hybrid system Reporter gene Bait protein DNA Binding Page 255

The yeast two-hybrid system Reporter gene Prey protein DNA activation Prey protein DNA activation Prey protein DNA activation Prey protein DNA activation Page 255

The yeast two-hybrid system Reporter gene Bait protein DNA Binding Prey protein DNA activation Page 255

The yeast two-hybrid system Reporter gene Bait protein DNA Binding Prey protein DNA activation Isolate and sequence the cDNA of the binding partner you have found Page 255

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 Page 258 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.

Yeast topoisomerase II E. coli gyrase B E. coli gyrase A The Rosetta Stone approach Page 258

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

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