Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes
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...
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Published in | The Journal of cell biology Vol. 183; no. 2; pp. 223 - 239 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
The Rockefeller University Press
20.10.2008
Rockefeller University Press |
Subjects | |
Online Access | Get full text |
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Summary: | 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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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 PMCID: PMC2568020 Correspondence to Laura Trinkle-Mulcahy: ltrinkle@uottawa.ca; or Angus Lamond: angus@lifesci.dundee.ac.uk Abbreviations used in this paper: FP, fluorescent protein; SILAC, stable isotope labeling with amino acids in cell culture. |
ISSN: | 0021-9525 1540-8140 |
DOI: | 10.1083/jcb.200805092 |