1. Announcements: Proposal resubmissions are due 4/23. It is recommended that students set up a meeting to discuss modifications for the final step of the assignment.

2. The Proteome Using high-throughput methods to identify proteins and to understand their function

3. What is proteomics? An organism’s proteome –A catalog of all proteins Expressed throughout life Expressed under all conditions The goals of proteomics –To catalog all proteins –To understand their functions –To understand how they interact with each other

4. Protein-protein interactions “The Interactome” Yeast two-hybrid analysis Other “protein complementation” methods Biochemical purification/Mass spectrometry

5. 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

6. TAP Purification Strategy

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

8. 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

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

10. Principle of mass spectrometry

11. 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

12. 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

13. A mass spectrum

14. 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

15. 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

22. Comparison of Fly Interactome Studies Yeast Two HybridcoAffinity Purification/Mass Spec Pub’d 2003Pub’d 2011 Done in YeastDone in S2 cells (embryo hemocytes) 10,623 clones examined3,488 clones examined 20,405 total interactions209,912 total interactions 7,048 proteins involved in total4,927 proteins involved in total 4,780 high confidence interactions10,969 high confidence interactions* 4,579 proteins in hci2,297 proteins in hci 586 proteins w/ no previous function *used different model for confidence

23. Summary I Goals of proteomics –Identify and ascribe function to proteins under all biologically plausible conditions Some accomplishments of proteomics –For yeast and flies Yeast two-hybrid method reveals interactome Subcellular localization of proteins Long way yet to accomplish overall goals