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 Fusion protein Chromosome PCR product COOH NH 2 Homologous recombination GFP HIS3MX6 ORF1 ORF2 protein GFP

4. FlyTrap Screen for Protein Localization http://flytrap.med.y ale.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 protein 1 to DNA-binding domain (bait) –Attach protein 2 to activation domain (prey) –Reporter gene expressed only if protein 1 and protein 2 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: Flies: Screened 10,000 predicted transcripts Screened 10,000 predicted transcripts Found 20,000 interactions Found 20,000 interactions Statistically assigned 4800 as “high quality” 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 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 ESI MALDI

21. Mass analyzers used for proteomics 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 Ion Trap 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