Proteomic analysis of membrane and membrane raft proteins is complicated by their inherent insolubility, which exacerbates difficulties with in-solution digestion of the proteins prior to ESI-LC-MS/MS. In-gel digestion yields more comprehensive proteomic and protein coverage of membrane/membrane raft samples, for example by LC-MS/MS of protein samples resolved by 1D SDS-polyacrylamide gel electrophoresis. Although this type of analysis can be performed quantitatively by labelling at the protein level, for instance by SILAC, the separation of proteins on a resolving gel complicates the application of other quantitative methods that employ post-digestion labelling techniques. This chapter describes an alternative protocol to prepare membrane or membrane raft protein samples to be isolated, but not separated, as unresolved bands in a gel. Focusing as a single band enables the confident excision of different samples in their entirety, to be digested, labelled, and fractionated for quantitative mass spectrometric analysis.
CITATION STYLE
Thompson, A. J., & Williamson, R. (2010). Protocol for quantitative proteomics of cellular membranes and membrane rafts. Methods in Molecular Biology (Clifton, N.J.), 658, 235–253. https://doi.org/10.1007/978-1-60761-780-8_14
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