Mass spectrometry enables the unbiased characterization of protein complexes. The success of this approach and the amount of information that can be retrieved are highly dependent on the achieved purity of the protein complex to be analyzed. Here we describe a modified tandem affinity purification (moTAP) approach which can be used to isolate the tumor necrosis factor receptor 1 signaling complex for subsequent analysis by liquid chromatography-tandem mass spectrometry. Indeed, this approach can easily be adapted to the isolation of other membrane-bound and intracellular signaling complexes. This methodology allows for a highly sensitive analysis and characterization of complex components, including posttranslational modifications and for the identification of novel complex components.
Keywords: Immunoprecipitation; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Proteomics; TNF receptor 1 signaling complex (TNFR1-SC); Tandem affinity purification; Tumor necrosis factor (TNF).