GSAT501 - proteomics Name, home-town Students – previous lab experience –Lab you hope to end up in? Teachers – what is your current project

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

Schedule Monday morning (09:00 to ~12:00): –Talk – Introductions, intro to proteomics - Leonard –Talk – Sample prep & fractionation – Amanda –Talk – LC & Instrumentation - Nestor Monday afternoon (13:00 to 16:00): –Talk – Fragmentation & mass analyzers – Nick –Talk – Peptide ID – Nestor –Talk – Quantitation proteomics – Leonard

Schedule Tuesday morning (09:00 to 12:00): –Tutorial – LC operations – Nick –Tutorial – MALDI – Jason –Tutorial – Manual sequencing – Jason Tuesday afternoon (13:00 to 16:00): –Talk – Practical phosphoproteomics – Lindsay –Lab – In solution digestion – Lindsay

Schedule Wednesday morning (09:00 to 12:00): –Lab – Stage-tipping and phosphopeptide enrichment – Lindsay Wednesday afternoon (13:00 to 16:00): –Lab – Phosphopeptide enrichment – Lindsay –Lab – Load samples on LC-MS/MS – Lindsay & Nik

Schedule Thursday morning (09:00 to 12:00): –Talk – Protein complexes – Nick –Talk – In vivo proteomics – Anna –Talk – Degradomics – Theo Thursday afternoon (13:00 to 16:00): –Dry lab – Raw data & QC – Lindsay –Dry lab – Mascot – Ali –Dry lab – MaxQuant – Lindsay

Schedule Friday morning (09:00 to 12:00): –Talk – Ubiquitin – Thibault –Talk – Atypical peptides – Charlie –Talk – Future of proteomics – Leonard Friday afternoon (13:00 to 16:00): –Dry lab – Perseus & assignment – Lindsay & Nat

Overview - practical Biochemistry –Phosphoproteomics samples –Work in pairs Mass spec Bioinformatics –Database searching (Mascot) –Quantitation of data (MaxQuant)

End-of-week assignment Conference abstract 300 words 3 separate sections –Background –Results –Conclusion

Other expectation ASK LOTS OF QUESTIONS Feedback –New format – practical info

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

Modificomics Metabolomics Central dogma of ‘omics GenomicsFunctional genomics Proteomics

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

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

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 Mass spectrometry proteomics

2D gel electrophoresis

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

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

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