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In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.


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Authors

Rees, Johanna S 
Lowe, Nick 
Armean, Irina M 
Roote, John 
Johnson, Glynnis 

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.

Description

Keywords

Animals, Chromatography, Affinity, Drosophila Proteins, Drosophila melanogaster, Larva, Protein Binding, Protein Interaction Mapping, Protein Phosphatase 1, Proteome, Recombinant Fusion Proteins, Reproducibility of Results, Tandem Mass Spectrometry

Journal Title

Mol Cell Proteomics

Conference Name

Journal ISSN

1535-9476
1535-9484

Volume Title

10

Publisher

Elsevier BV
Sponsorship
Cancer Research Uk (None)
Wellcome Trust (092096/Z/10/Z)
Wellcome Trust (080007/Z/06/Z)
Wellcome Trust (076739/Z/05/Z)
This project is funded by the Welcome Trust.