Transcript Proteomics
Protein-Protein Interactions • High-throughput strategy – Prediction from sequence • In silico analysis – Protein A from species A: domain 1 and 2 – Protein 1’ and protein 2’ from species B • Recognition sequence homology – Yeast two-hybrid screen of whole genome – Tagged protein • Tandem affinity purification (TAP) + MS • Immunoprecipitation + MS – Ab to target proteins – Pooling assay • Biochemical functional assay 1 http://depts.washington.edu./sfields/ Y2H: basic design • If the two proteins interact, the reporter gene (here: HIS3) is switched on and the diploids can grow on -His plates: • If the two proteins don't interact, the reporter gene remains inactive and the cells can't grow on -His plates: 2 Discovery Q • Y2H protein interaction inside nucleus of yeast cell. Is it OK? • What is the proper control? • Is it restricted to yeast proteins only? 3 Large-scale Y2H • Yeast genome – Array screening • Much more time- and labor- intensive • More positive identification – Library screening • Reasonable time and effort – Bioinfomatics platform • http://portal.curagen.com Uetz, et al., Nature 2000 403, 623-627. Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261. 4 DIP search • Database of interacting proteins Start/root node 1st shell nodes 2nd shell nodes Interactions Color: reliability Width: no. of exp. http://dip.doe-mbi.ucla.edu/ 5 Protein network • Built by association Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261. 6 Interaction between groups • Crosstalk between and within functional groups Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261. 7 By location • Grouped by cellular compartments Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261. 8 Prediction of functions • Guilty-by-association Ypt1 Akr2 Yip1 YPL246C YHR105W YGL161C Vam7 Endocytosis Pep12 Vesicle transport & membrane fusion Schwikowski et al., 2000 Nature Biotech. 18, 1257-1261. 9 Tag-protein + MS • Co-precipitation – Tandem-affinity purification – SDS-PAGE • Mass spectrometry • Bioinformatics Kumar and Snyder, 2002 Nature 415, 123-124 10 Pooling functional assay • Biochemical assay for activity – 6144 GST-ORF strains – 64 pools of 96 fusions/plate each – Pools of 12 columns and 8 columns 1 12 1 8 11 Priori and Potentials • Priori – The GST-ORF is functional – Soluble after extraction – Remain functional • Retains other required components when purified • Fast and sensitive • Potentials – Determine the range of the substrate proteins – Identifying gene leads to the binding of particular molecule, ligand, or drug. 12 Next Week • Please read Text p. 183-187. – 1:30 pm at room A105 – Advanced handout? • Paper discussion – Whatever we did not finish today • Homework assignment – How far did you get? 13