1. TAP(Tandem Affinity Purification) Billy Baader Genetics 677

2. Protein-protein interactions Protein Identification 20,000+ genes in humans Millions of proteins Understanding human biological processes

3. Protein Identification Other available methods -2D gel analysis -Labeling methods -Antibodies -Peptide tagging -Mass Spectrometry What are some of the problems with these methods?

4. Problems with Classical Methods Requires large amounts of protein Limitations in the number of testable samples Purification Contamination Time

5. How TAP works

8. TAP compared to other methods Flag Tag –Small peptide tag Natural protein levels vs. overexpressed proteins Yeast Two Hybrid –Low level of overlap –Assays protein interactions

9. Protein internal structure Desire to understand molecular mechanisms Some proteins lack obvious enzymatic activity Once proteins are discovered we would like to know how they work and interact Possibilities -Crystillization -Electron Microscopy -Two Hybrid Assay -Chemical Cross-Linking

10. Functional organization of the yeast proteome by systematic analysis of protein complexes Gavin et al. Practical application of TAP and mass spectrometry on S. cerevisiae Emphasizes the potential for a massive amount of information to be obtained through TAP

11. Potential of protein knowledge “Whenever it has been possible to retrieve and analyze particular cellular protein complexes under physiological conditions, the insight gained from the analysis has been fundamental for the biological understanding of their function” i.e. spliceosome, cyclosome, proteasome Examined 25% of ORFs in yeast

12. Method for purification TAP 1. High affinity purification 2. Elution 3. Second affinity purification Separate with gel electrophoresis Digest with trypsin Analyze with mass spectrometry

13. TAP tags TAP cassette created through PCR Insertion at the C-terminus of a selected yeast ORF by homologous recombination Examined 1,739 genes

14. Homologous Recombination of TAP tag

15. Proteins purified from different organelles

16. Efficiency

17. Examining the data Technical Bias against proteins below 15kDa Possiblity of using different entry points to purify protein complexes Comparison to literature 70% reproducability

18. Polyadenylation machinery Responsible for eukaryotic mRNA cleavage and polyadenylation Single entry point used: Pta1 12 of 13 known interactors and 7 new components

19. Reproducibility using various entry points

20. Polyadenylation machinery

21. Protein complex networks Utilized an algorithm to automatically generate map Links are between complexes sharing at least one protein

22. Protein Network

23. Orthologs Examined the hypothesis that orthologous gene products are responsible for essential cellular activities Orthologous complexes interact preferentially with other orthologous complexes Nonorthologous complexes do not interact at as well with the orthologous complexes The same relation is present between essential and non-essential complexes

24. Human/Yeast Orthologs Arp2/3 –Cytoskeleton-associated complex Ccr4-Not –Involved in control of gene expression TRAPP –Transport protein particle –associated with Golgi body

25. Ortholog Comparison Results

26. Results Huge increase in number of proteome components TAP was responsible for a efficient identificaiton of low-abundance proteins as well as large complexes Differences in the aspects of protein interaction detected through TAP compared with Y2H Orthologous complexes appear to represent the building blocks of a ‘core proteome’

27. Advantages of TAP Simplicity Cleaner, more intact complexes Higher yield Low false negative rates Tags show little protein alteration

28. TAP vs. Y2H Analyzes complexes and can create protein network maps Analyzes more of the proteome Works in membrane proteins Works in many organisms Analyzes binary interaction between proteins Works with transient protein interactions Performed in vivo These methods yield different information and should be used complementarily

29. Possibilities Gavin et al. believe that there methods are one of the most efficient and unambiguous routes towards the assignment of gene identity and function Easy to analyze large amounts of protein complexes and assess there relation to each other Increase in understanding of biological systems and their processes Drug discovery and usage may be greatly enhanced through this knowledge Identification of a vast number of proteins and protein complexes Other techniques may be used to understand the function of these proteins A more complete understanding of the proteomes can hopefully be developed

30. Questions The review, when talking about TAP, said that it has been used to purify membrane bound protein complexes. I don't know a lot about protein purification, but I have always heard that purifying membrane proteins is notoriously difficult. Can you explain what make TAP better suited for this than other methods?

31. Questions In the Review paper table 1 compares Flag and TAP; why is the fraction of successful purification (both with and without interacting proteins) higher for Flag? The other statistics in the table seem to make Flag a poor alternative, however, the fraction of successful purifications would seem to be an important percentage to raise. What is being done to improve this?

32. Questions