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Citation
Rees, J.S., Lowe, N., Armean, I.M., Roote, J., Johnson, G., Drummond, E., Spriggs, H., Ryder, E., Russell, S., Johnston, D.S., Lilley, K.S. (2011). In Vivo Analysis of Proteomes and Interactomes Using Parallel Affinity Capture (iPAC) Coupled to Mass Spectrometry.  Mol. Cell. Proteomics 10(6): M110.002386.
FlyBase ID
FBrf0213845
Publication Type
Research paper
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.

PubMed ID
PubMed Central ID
PMC3108830 (PMC) (EuropePMC)
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Secondary IDs
    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Mol. Cell. Proteomics
    Title
    Molecular and Cellular Proteomics
    Publication Year
    2002-
    ISBN/ISSN
    1535-9476
    Data From Reference