Application of I-DIRT technology for protein interaction studies in D. melanogaster embryos

摘要

SILAC has revolutionized analysis of protein expression levels and protein complex formation (Mann, 2006). One isotopic technique developed in S. cerevisiae, I-DIRT (isotopic differentiation of interactions as random or targeted), provides for unambiguous identification of 'real' and 'contaminant' protein interactions during protein complex immunoisolations (Tackett, 2005). I-DIRT is performed by mixing isotopically heavy untagged cells with isotopically light affinity-tagged cells, co-lysis, immunoisolation and MS to determine 'real' (isotopically light) from 'contaminant' (1 light: 1 heavy) associating proteins. We describe the methodology to transpose this technique to Drosophila melanogaster, to identify interactions with Pipsqueak (Psq). Psq is an important developmental regulator - having pleiotropic functions during oogenesis, embryogenesis, and adult development (Lehmann, 1998).