Analyzing Protein-Protein Interactions by Quantitative Mass Spectrometry

Methods. 2011 Aug;54(4):387-95. doi: 10.1016/j.ymeth.2011.03.001. Epub 2011 Mar 5.

Abstract

Since most cellular processes depend on interactions between proteins, information about protein-protein interactions (PPIs) provide valuable insights into protein function. Over the last years, quantitative affinity purification followed by mass spectrometry (q-AP-MS) has become a powerful approach to investigate PPIs in an unbiased manner. In q-AP-MS the protein of interest is biochemically enriched together with its interaction partners. In parallel, a control experiment is performed to control for non-specific binding. Quantitative mass spectrometry is then employed to compare protein levels in both samples and to exclude non-specific contaminants. Here, we provide two detailed q-AP-MS protocols for pull-downs with immobilized bait proteins or transient transfection of tagged expression constructs. We discuss benefits and limitations of q-AP-MS and highlight critical parameters that need to be considered. The protocols and background information presented here allow the reader to adapt the generic q-AP-MS strategy for a wide range of biological questions.

Publication types

  • Review

MeSH terms

  • Chromatography, Liquid / methods
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Isotope Labeling
  • Protein Interaction Mapping / methods*
  • Proteins / chemistry*
  • Tandem Mass Spectrometry / methods*

Substances

  • Proteins