In Vivo Analysis of Proteomes and Interactomes Using Parallel Affinity Capture (iPAC) Coupled to Mass Spectrometry

• DOI: 10.1074/mcp.m110.002386
• Publication Date: 2011-03-30T02:41:47Z
• Authors: Johanna S. Rees; Nick Lowe; Irina M. Armean; John Roote; Glynnis Johnson; Emma Drummond; Helen Spriggs; Edward Ryder; Steven Russell; Daniel St Johnston; Kathryn S. Lilley
• Keywords: Proteome

Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster triple tagged with Flag, Strep II, Yellow fluorescent protein in vivo within affinity pull-down experiments isolated these native complexes from embryos. We describe pipeline determining interactomes by Parallel Capture (iPAC) show its use partners of several baits range sizes subcellular locations. This protocol employs the different tags parallel involves detailed comparison resulting data sets, ensuring interaction lists achieved are high confidence. that approach identifies known interactors bait as well novel comparing published sets. The confidence sets presented here add new currently incomplete D. interactome. Additionally report contaminant persistent purifications irrespective bait.