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Genomics II: The Proteome

Genomics II: The Proteome. Using high-throughput methods to identify proteins and to understand their function. Subcellular localization of the yeast proteome. Complete genome sequences allow each ORF to be precisely tagged with a reporter molecule

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Genomics II: The Proteome

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  1. Genomics II:The Proteome Using high-throughput methods to identify proteins and to understand their function

  2. 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

  3. Attaching a GFP tag to an ORF GFP HIS3MX6 PCR product Homologous recombination Chromosome ORF2 ORF1 protein COOH NH2 GFP Fusion protein

  4. FlyTrap Screen for Protein Localization http://flytrap.med.yale.edu/

  5. Patterns of protein localization

  6. Distribution of subcellular localization

  7. Identification of unpredicted ORFs

  8. Protein-protein interactions“The Interactome” • Yeast two-hybrid analysis • Protein chips • Biochemical purification/Mass spectrometry • Protein complementation

  9. Yeast two-hybrid method • Goal: Determine how proteins interact with each other • Method • Use yeast transcription factors • Gene expression requires the following: • A DNA-binding domain • An activation domain • A basic transcription apparatus • Attach protein1 to DNA-binding domain (bait) • Attach protein2 to activation domain (prey) • Reporter gene expressed only if protein1 and protein2 interact with each other

  10. A schematic of the yeast two-hybrid method m n

  11. Results from a yeast two-hybrid experiment • Goal: To characterize protein–protein interactions among 6,144 yeast ORFs • 5,345 were successfully cloned into yeast as both bait and prey • Identity of ORFs determined by DNA sequencing in hybrid yeast • 692 protein–protein interaction pairs • Interactions involved 817 ORFs

  12. Yeast two-hybrid results for flies & worms • Worms: • Created >3000 bait constructs • Tested against two AD libraries • Mapped 4000 interactions • Flies: • Screened 10,000 predicted transcripts • Found 20,000 interactions • Statistically assigned 4800 as “high quality” interactions

  13. Caveats associated with the yeast two-hybrid method • There is evidence that other methods may be more sensitive • Some inaccuracy reported when compared against known protein–protein interactions • False positives • False negatives

  14. Purification of interacting proteins • Immunoprecipitation • Impractical on large scale (identification of unknowns) • Affinity purification • Biochemically practical, but too dirty • Tandem affinity purification • Sufficient yield & purity for identification of unknown proteins

  15. TAP Purification Strategy

  16. Identification of Interacting Proteins Proteolytic Digestion (Trypsin) Mass Spectrometric Analysis

  17. Identifying proteins with mass spectrometry • Preparation of protein sample • Extraction from a gel • Digestion by proteases — e.g., trypsin • Mass spectrometer measures mass-charge ratio of peptide fragments • Identified peptides are compared with database • Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database • Match of experimental readout to database PMF allows researchers to identify the protein

  18. Mass spectrometry • Measures mass-to-charge ratio • Components of mass spectrometer • Ion source • Mass analyzer • Ion detector • Data acquisition unit A mass spectrometer

  19. Principle of mass spectrometry

  20. Ion sources used for proteomics ESI • Proteomics requires specialized ion sources • Electrospray Ionization (ESI) • With capillary electrophoresis and liquid chromatography • Matrix-assisted laser desorption/ionization (MALDI) • Extracts ions from sample surface MALDI

  21. Mass analyzers used for proteomics Ion Trap • Ion trap • Captures ions on the basis of mass-to-charge ratio • Often used with ESI • Time of flight (TOF) • Time for accelerated ion to reach detector indicates mass-to-charge ratio • Frequently used with MALDI • Also other possibilities Time of Flight Detector

  22. A mass spectrum

  23. Identifying proteins with mass spectrometry • Preparation of protein sample • Extraction from a gel • Digestion by proteases — e.g., trypsin • Mass spectrometer measures mass-charge ratio of peptide fragments • Identified peptides are compared with database • Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database • Match of experimental readout to database PMF allows researchers to identify the protein

  24. Limitations of mass spectrometry • Not very good at identifying minute quantities of protein • Trouble dealing with phosphorylated proteins • Doesn’t provide concentrations of proteins • Improved software eliminating human analysis is necessary for high-throughput projects

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