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

Analyzing protein-protein interactions by quantitative mass spectrometry.

Author information

1
Cell Signaling and Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

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.

PMID:
21382495
DOI:
10.1016/j.ymeth.2011.03.001
[Indexed for MEDLINE]

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