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Article Dans Une Revue Journal of Cell Biology Année : 2008

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

Séverine Boulon
François-Michel Boisvert
  • Fonction : Auteur
Yun Wah Lam
  • Fonction : Auteur
Laura Trinkle-Mulcahy
  • Fonction : Auteur
Séverine Boulon
  • Fonction : Auteur
Yun Wah Lam
  • Fonction : Auteur
Roby Urcia
  • Fonction : Auteur
François-Michel Boisvert
  • Fonction : Auteur
Nick A Morrice
  • Fonction : Auteur
Sam Swift
  • Fonction : Auteur
Ulrich Rothbauer
  • Fonction : Auteur
Heinrich Leonhardt
  • Fonction : Auteur
Angus Lamond
  • Fonction : Auteur

Résumé

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.
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Dates et versions

hal-03030167 , version 1 (29-11-2020)

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Séverine Boulon, François-Michel Boisvert, Yun Wah Lam, Laura Trinkle-Mulcahy, Séverine Boulon, et al.. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. Journal of Cell Biology, 2008, 183 (2), pp.223-239. ⟨10.1083/jcb.200805092⟩. ⟨hal-03030167⟩
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