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

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