Analyzing protein-protein interactions by quantitative mass spectrometry

Cell Signaling and Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Methods (Impact Factor: 3.65). 03/2011; 54(4):387-95. DOI: 10.1016/j.ymeth.2011.03.001
Source: PubMed


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.

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    • "Proteins often functionally interact as part of complex and dynamic networks [1] [2]. An important goal of current proteomics research is to develop techniques that can accurately identify such protein 'interactomes' [3]. A popular method uses epitope-tagged proteins in pull-down assays. "
    Dataset: SILAC-iPAC
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    • "We performed immuno-based affinity purification experiments followed by mass spectrometric protein identification (46) using the E13-inducible ES clones with the 3xFLAG (‘Materials and Methods’ section). We identified 23 potential protein partners of E13 with high-confidence (Table 2), among which there are two TFs [Gtf2e2, Btf3 (47)], several mRNA processing proteins and two components of the Polycomb complex [Eed, Suz12 (48) and the retinoblastoma binding protein Rbbp4, which may be involved in pluripotent stem cell maintenance and neuronal differentiation (49)]. "
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