Protocol for Quantitative Proteomics of Cellular Membranes and Membrane Rafts
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.
Key wordsQuantitative proteomics membrane raft Isobaric tagging in-gel digestion strong cation exchange chromatography LC-MS/MS
The authors would like to thank Dr. Amy Pooler and Dr. Helen Byers for advice and assistance with reviewing the protocols. This work was funded by the Medical Research Council and the Department of Trade and Industry, UK.
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