title: In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.
abstract: 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 proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.
authors: Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK.
authors: Johanna S
authors: JS
authors: Rees
authors: Nick
authors: N
authors: Lowe
authors: Irina M
authors: IM
authors: Armean
authors: John
authors: J
authors: Roote
authors: Glynnis
authors: G
authors: Johnson
authors: Emma
authors: E
authors: Drummond
authors: Helen
authors: H
authors: Spriggs
authors: Edward
authors: E
authors: Ryder
authors: Steven
authors: S
authors: Russell
authors: Daniel
authors: D
authors: St Johnston
authors: Kathryn S
authors: KS
authors: Lilley
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D029721:Drosophila Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D007814:Larva / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D054645:Protein Phosphatase 1 / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D029721:Drosophila Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D054645:Protein Phosphatase 1