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Genomics II: The Proteome. Using high-throughput methods to identify proteins and to understand their function. Contents. Definition of proteomics Protein profiling 2-D gel electrophoresis Protein chips Protein-protein interactions Yeast two-hybrid method Protein chips
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Genomics II:The Proteome Using high-throughput methods to identify proteins and to understand their function
Contents • Definition of proteomics • Protein profiling • 2-D gel electrophoresis • Protein chips • Protein-protein interactions • Yeast two-hybrid method • Protein chips • TAP tagging/Mass spectrometry • Biochemical genomics • Using proteomics to uncover transcriptional networks
What is proteomics? • An organism’s proteome • A catalog of all proteins • Expressed throughout life • Expressed under all conditions • The goals of proteomics • To catalog all proteins • To understand their functions • To understand how they interact with each other
The challenges of proteomics • Splice variants create an enormous diversity of proteins • ~25,000 genes in humans give rise to 200,000 to 2,000,000 different proteins • Splice variants may have very diverse functions • Proteins expressed in an organism will vary according to age, health, tissue, and environmental stimuli • Proteomics requires a broader range of technologies than genomics
Diversity of function in splice variants • Example: the calcitonin gene • Gene variant #1 • Protein: calcitonin • Function: increases calcium uptake in bones • Gene variant #2 • Protein: calcitonin gene-related polypeptide • Function: causes blood vessels to dilate
Posttranslational modifications • Proteolytic cleavage • Fragmenting protein • Addition of chemical groups
Chemical modifications • Phosphorylation: activation and inactivation of enzymes • Acetylation: protein stability, used in histones • Methylation: regulation of gene expression • Acylation: membrane tethering, targeting • Glycosylation: cell–cell recognition, signaling • GPI anchor: membrane tethering • Hydroxyproline: protein stability, ligand interactions • Sulfation: protein–protein and ligand interactions • Disulfide-bondformation: protein stability • Deamidation: protein–protein and ligand interactions • Pyroglutamicacid: protein stability • Ubiquitination: destruction signal • Nitration of tyrosine: inflammation
Protein Profiling:Practical applications • Comparison of protein expression in diseased and normal tissues • Likely to reveal new drug targets • Today ~500 drug targets • Estimates of possible drug targets: 10,000–20,000 • Protein expression signatures associated with drug toxicity • To make clinical trials more efficient • To make drug treatments more effective
2-D gel electrophoresis Acidic Basic • Polyacrylamide gel • Voltage across both axes • pH gradient along first axis neutralizes charged proteins at different places • pH constant on a second axis where proteins are separated by weight • x–y position of proteins on stained gel uniquely identifies the proteins High MW Low MW
Differential in gel electrophoresis with benzoic acid Cy3 • Label protein samples from control and experimental tissues • Fluorescent dye #1 for control • Fluorescent dye #2 for experimental sample • Mix protein samples together • Identify identical proteins from different samples by dye color without benzoic acid Cy5
Caveats associated with 2-D gels • Poor performance of 2-D gels for the following: • Very large proteins • Very small proteins • Less abundant proteins • Membrane-bound proteins • Presumably, the most promising drug targets
Protein chips • Thousands of proteins analyzed simultaneously • Wide variety of assays • Antibody–antigen • Enzyme–substrate • Protein–small molecule • Protein–nucleic acid • Protein–protein • Protein–lipid Yeast proteins detected using antibodies
Subcellular localization of the yeast proteome • Complete genome sequences allow each ORF to be precisely tagged with a reporter molecule • Tagged ORF proteins indicate subcellular localization • Useful for the following: • Correlating to regulatory modules • Verifying data on protein–protein interactions • Annotating genome sequence
Attaching a GFP tag to an ORF GFP HIS3MX6 PCR product Homologous recombination Chromosome ORF2 ORF1 protein COOH NH2 GFP Fusion protein
Location of proteins revealed cytoplasm • 75% of yeast proteome localized • > 40% of proteins in cytoplasm • 67% of proteins were previously unlocalized • Localizations correlate with transcriptional modules nucleus A protein localized to the nucleus
FlyTrap Screen for Protein Localization http://flytrap.med.yale.edu/
