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.
Thompson
Andrew J
AJ
MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, London, UK.
Williamson
Ritchie
R
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D008565:Membrane Proteins; C106354:flotillins