Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.
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.
Veraksa
Alexey
A
Massachusetts General Hospital Cancer Center, Department of Cell Biology, Harvard Medical School, Charlestown, Massachusetts 02129, USA. veraksa@helix.mgh.harvard.edu
Bauer
Andreas
A
Artavanis-Tsakonas
Spyros
S
D000345:Affinity Labels; D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D004330:Drosophila / Q000166:cytology / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000032:analysis* / Q000302:isolation & purification*; D004625:Embryo, Nonmammalian; D005822:Genetic Vectors; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis; D040901:Proteomics; D051880:Receptors, Notch; D015398:Signal Transduction
D000345:Affinity Labels; D029721:Drosophila Proteins; D008565:Membrane Proteins; C497759:N protein, Drosophila; D020543:Proteome; D051880:Receptors, Notch