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

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

352 Scopus Citations
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Author(s)

  • Laura Trinkle-Mulcahy
  • Séverine Boulon
  • Yun Wah Lam
  • Roby Urcia
  • Francois-Michel Boisvert
  • And 6 others
  • Franck Vandermoere
  • Nick A. Morrice
  • Sam Swift
  • Ulrich Rothbauer
  • Heinrich Leonhardt
  • Angus Lamond

Detail(s)

Original languageEnglish
Pages (from-to)223-239
Journal / PublicationJournal of Cell Biology
Volume183
Issue number2
Publication statusPublished - 20 Oct 2008

Link(s)

Abstract

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 non-specific 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.

Research Area(s)

Citation Format(s)

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. / Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, Francois-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus.

In: Journal of Cell Biology, Vol. 183, No. 2, 20.10.2008, p. 223-239.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

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