Abstract
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.
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Acknowledgments
We thank P. Draber and L. Spilgies for providing crucial technical advice and L. Spilgies for providing western blot stainings. This work was supported by a Wellcome Trust Senior Investigator Award (096831/Z/11/Z), an ERC Advanced Grant (294880), and a Cancer Research UK programme grant (A17341) awarded to H.W.; and a BBSRC CASE studentship (BB/J013129/1) awarded to M.R. This work was also supported by the CRUK–UCL Centre grant (515818) and the Cancer Immunotherapy Accelerator (CITA) Award (525877) from CRUK.
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Reichert, M., Bhamra, A., Kupka, S., Walczak, H. (2018). Characterization of the TNFR1-SC Using “Modified Tandem Affinity Purification” in Conjunction with Liquid Chromatography–Mass Spectrometry (LC-MS). In: Ting, A. (eds) Programmed Necrosis. Methods in Molecular Biology, vol 1857. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8754-2_16
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DOI: https://doi.org/10.1007/978-1-4939-8754-2_16
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