scholarly journals Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry

2010 ◽  
Vol 9 (8) ◽  
pp. 1650-1665 ◽  
Author(s):  
Robyn M. Kaake ◽  
Xiaorong Wang ◽  
Lan Huang
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein Interaction Networks ◽  
Interaction Networks ◽  
Quantitative Mass Spectrometry

scholarly journals Deciphering Protein Complexes and Protein Interaction Networks by Tandem Affinity Purification and Mass Spectrometry

2002 ◽  
Vol 1 (3) ◽  
pp. 204-212 ◽  
Author(s):  
Anna Shevchenko ◽  
Daniel Schaft ◽  
Assen Roguev ◽  
W. W. M. Pim Pijnappel ◽  
A. Francis Stewart ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Protein Interaction Networks ◽  
Interaction Networks

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

2008 ◽  
Vol 183 (2) ◽  
pp. 223-239 ◽  
Author(s):  
Laura Trinkle-Mulcahy ◽  
Séverine Boulon ◽  
Yun Wah Lam ◽  
Roby Urcia ◽  
François-Michel Boisvert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Cell Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Specific Protein ◽  
Protein Interaction Data ◽  
Quantitative Mass Spectrometry ◽  
Cell Extracts ◽  
Interaction Partners

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.

Sign in / Sign up

Export Citation Format

Share Document