Analyzing protein complexes in Drosophila with tandem affinity purification–mass spectrometry
2005; Wiley; Volume: 232; Issue: 3 Linguagem: Inglês
10.1002/dvdy.20272
ISSN1097-0177
AutoresAlexey Veraksa, Andreas Bauer, Spyros Artavanis‐Tsakonas,
Tópico(s)Ubiquitin and proteasome pathways
ResumoAbstract We describe the application of tandem affinity purification–mass spectrometry (TAP‐MS) to the study of protein complexes in Drosophila . We have constructed vectors for inducible expression of TAP‐tagged fusion proteins in Drosophila cultured cells and in vivo. Using these vectors, we tagged, as a paradigm, several components of the Notch signaling pathway, isolated protein complexes containing the baits and associated proteins from cells and embryos, and identified the subunits by liquid chromatography–tandem mass spectrometry (LC‐MS/MS). Several known interactions involving Notch pathway elements were confirmed, and many novel potential interactions were uncovered. For some of the novel associations, we validated the interaction genetically and biochemically. We conclude that TAP, in combination with MS, can be used as an effective method for the studies of the Drosophila proteome. Developmental Dynamics 232:827–834, 2005. © 2005 Wiley‐Liss, Inc.
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