GSAT501 - proteomics

1. GSAT501 - proteomics • Name, home-town • Students – previous lab experience • Teachers – what is your current project

2. Overview - theory • Instrumentation • Quantitation • Protein identification (informatics) • Experimental design • Applications

3. Schedule • Monday morning (09:30 to ~12:00): • Talk – Introductions, intro to proteomics - Leonard • Talk – Ionization – Vince • Monday afternoon (12:45 to 16:30): • Talk – Mass analyzers – Vince • Talk – Practical applications – Marta • Talk – Fragmentation – Nick

4. Schedule • Tuesday morning (09:00 to 12:30): • Talk – Peptide sequencing – Nick • Dry lab – Manually sequence spectra – Jason • Tuesday afternoon (13:30 to 16:00): • Talk – Practical proteomics – Sarah • Talk – Quantitative proteomics – Leonard

5. Schedule • Wednesday morning (09:00 to 14:15): • Lab – In solution digestion – Jenny • Lab – In gel digestion – Suzanne/Jamie • Two overlapping exercises • Wednesday afternoon (14:15– 16:30): • Talk – Practical applications – Thibault • Talk – Fractionation procedures – Sarah

6. Schedule • Thursday morning (09:00 to 12:30): • Tutorial – MALDI – Suzanne • Tutorial – LC operations – Nick • Lab – STAGE tipping – Suzanne/Jamie • Thursday afternoon (13:15 to 16:30): • Lab – Dimethyl labelling – Jenny • Lab – Run samples overnight – Nick/Sarah

7. Schedule • Friday morning (09:00 to 12:00): • Talk – Database searching – Sarah • Lab – Mascot – Jason • Friday afternoon (13:00 to 16:30+): • Lab – Proteome Discoverer – Jenny • Lab – MaxQuant – Anders • Talk – Practical applications – Anders • Beers…

8. Overview - practical • Two sets of samples • In-gel and in-solution digestion of proteins • Chemical derivatization with isotopes • Database searching (Mascot) • Quantitation of data (MaxQuant)

9. End-of-week assignment • Conference abstract • 300 words • 3 separate sections • Background • Results • Conclusion

10. Other expectation • ASK LOTS OF QUESTIONS • Feedback • New format

11. A proteome: • Strict: all the proteins expressed from a genome • Loose: the proteins expressed in a tissue or cell at a given time under a specific set of conditions • Looser: all the proteins in a sample (protein complex, structure, fluid or organelle

12. Genomics Functional genomics Proteomics Modificomics Metabolomics Central dogma of ‘omics

13. What is proteomics? • Study of all proteins in a cell, tissue or organism • Temporal, conditional • Mass spectrometry - identify & quantify • Protein chips - identify & quantify • Structural - function • Imaging - location • de Hoog & Mann article

14. Imaging proteomics • 25,000 genes in humans • ~10,000 antibodies available (all organisms) • Where are proteins expressed? • High-throughput cloning • High-throughput antibody generation • Rabbit and chicken • Stained tissues checked by pathologist

15. Mass spectrometry proteomics • Use of MS to identify, quantify and/or characterize ‘all’ proteins in a sample • Not use of MS to study proteins • Current technology can: • ID hundreds to thousands of proteins • Identify modifications on proteins • Current technology cannot: • ID all proteins in a sample • Characterize most modifications on an omic scale

16. 2D gel electrophoresis

17. 2D gel electrophoresis

18. Top-down vs. bottom-up • Mostly interested in proteins • Optimal mass ranges differ among instruments • Very low masses - accelerator MS • MDa - quadrupoles • Trade-off between mass and ionizability • Whole protein proteomics not ready for prime time

19. Proteins measured clinically in plasma span >10 orders of magnitude in abundance Leigh Anderson, PPI

20. Where field is going • Biomarkers • Post-translational modifications • Interactome • Complement genomics & answer other questions