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Keywords:

  • interactomics;
  • protein interactions;
  • affinity purification;
  • quantitative proteomics;
  • mass spectrometry;
  • stable isotope labeling;
  • SILAC

Abstract

Many physiological processes are regulated by dynamic protein interaction networks whose characterization provides valuable information on cell biology. Several strategies can be used to analyze protein–protein interactions. Among them, affinity purification combined with mass spectrometry (AP-MS) is arguably the most widely employed technique, not only owing to its high throughput and sensitivity but also because it can answer critical questions such as where, when, and how protein–protein interactions occur. In AP-MS workflows, both the target protein and its interacting partners are isolated before being identified by MS. The main challenge of this approach is to distinguish bona fide binders from background contaminants. This review focuses on the different strategies designed to circumvent this limitation. In this regard, the combination of quantitative proteomics and affinity purification emerges as one of the most powerful, yet relatively simple, strategies to characterize protein–protein interactions. © IUBMB Life, 65(1):9–16, 2013