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Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

¹ Wellcome Trust Centre for Gene Regulation and Expression, and ² MRC Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, UK
³ Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada K1H 8M5
⁴ Department of Biology and Chemistry, City University of Hong Kong, Hong Kong
⁵ Department of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK
⁶ Department of Biology, LMU Biozentrum, 82152 Planegg-Martinsried, Germany

Correspondence to Laura Trinkle-Mulcahy: ltrinkle{at}uottawa.ca; or Angus Lamond: angus{at}lifesci.dundee.ac.uk

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

© 2008 Trinkle-Mulcahy et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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