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

Laura Trinkle-Mulcahy (Lead / Corresponding author), Severine Boulon, Yun Wah Lam, Roby Urcia, Francois-Michel Boisvert, Franck Vandermoere, Nick A. Morrice, Sam Swift, Ulrich Rothbauer, Heinrich Leonhardt, Angus Lamond (Lead / Corresponding author)

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    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 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.

    Original languageEnglish
    Pages (from-to)223-239
    Number of pages17
    JournalJournal of Cell Biology
    Volume183
    Issue number2
    DOIs
    Publication statusPublished - 20 Oct 2008

    Keywords

    • MOTOR-NEURON COMPLEX
    • SMN COMPLEX
    • GENE-PRODUCT
    • CELL-CULTURE
    • AMINO-ACIDS
    • U7 SNRNPS
    • COMPONENT
    • SURVIVAL
    • IDENTIFICATION
    • FIBRILLARIN

    Cite this

    Trinkle-Mulcahy, L., Boulon, S., Lam, Y. W., Urcia, R., Boisvert, F-M., Vandermoere, F., Morrice, N. A., Swift, S., Rothbauer, U., Leonhardt, H., & Lamond, A. (2008). Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. Journal of Cell Biology, 183(2), 223-239. https://doi.org/10.1083/jcb.200805092