interexp - Experimental Techniques 2 High-throughput...

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Experimental Techniques 2
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High-throughput interaction detection Yeast two-hybrid - pairwise organisms as machines to learn about organisms yeast, worm, fly, human,... low intersection between repeated experiments in vivo , but takes place inside the nucleus. Estimated 50% FP rate TAP-MS (co-immunoprecipitation) - complexes
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Tandem Affinity Purification Glu-Asn-Leu-Tyr-Phe-Gln-Gly “Protein A” from Staphylococcus aureus Binds to IgG protein TEV protease cleavage (cutting) site Calmodulin binding peptide 3’ 5’ Want to find interaction partners for protein encoded by this gene: Add a tag to the end of its DNA sequence. (Puig et al, 2001) TOBACCO ETCH VIRUS PROTEASE
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Fishing for Proteins Contaminants Tagged protein Co-complexed proteins IgG IgG Calmodulin Tag may not be exposed Tag may change folding / binding properties Tag may change expression levels Grab with Immunoglobulin G protein Wash contaminants cleave with TEV Retrieve with calmodulin beads
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Sequencing Proteins (Tandem Mass Spectrometry) AAVEK A AA AAV AAVE AAVEK K EK VEK AVEK b-ions y-ions Tandem Mass Spec frequency of seeing given mass Database of known or predicted spectra Search Trypsin digestion
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Gavin et al, 2002 Results: 589 tagged proteins (78% of which returned some interaction partners) 232 complexes (grouping those with substantial overlap) Covering 1440 proteins Not binary interactions In this picture: edges mean complexes share a protein
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Gavin et al, 2006 - Larger scale TAP-MS: 2006 update: - 2,760 unique proteins involved in some complex (60% of the proteome of yeast) - Reproducible: repeated experiment for 139 proteins, and 69% of retrieved proteins were common to both experiments.
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