Proteomic Analysis of Detergent-Resistant Membrane Rafts
A combined, detergent- and organic solvent-based proteomic method for the analysis of detergent-resistant membrane rafts (DRMR) is described. These specialized domains of the plasma membrane contain a distinctive and dynamic protein and/or lipid complement, which can be isolated from most mammalian cells. Lipid rafts are predominantly involved in signal transduction and adapted to mediate and produce different cellular responses. To facilitate a better understanding of their biology and role, DRMR were isolated from Vero cells as a Triton X-100 insoluble fraction. After detergent removal, sonication in 60% buffered methanol was used to extract, solubilize and tryptically digest the resulting protein complement. The peptide digestate was analyzed by microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. Gas-phase fractionation in the mass-to-charge range was employed to broaden the selection of precursor ions and increase the number of identifications in an effort to detect less abundant proteins. A total of 380 proteins were identified including all known lipid raft markers. A total of 91 (24%) proteins were classified as integral alpha-helical membrane proteins, of which 51 (56%) were predicted to have multiple transmembrane domains.
Amino acid sequence, cell membrane, chlorocebus aethiops, detergents, gas chromatography-mass spectrometry, membrane, lipids, microdomains, proteins, octoxynol, vero cells
Blonder, Josip; Hale, Martha L.; Lucas, David A.; Schaefer, Carl F.; Yu, Li-Rong; Conrads, Thomas P.; Issaq, Haleem J.; Stiles, Bradley G.; and Veenstra, Timothy D., "Proteomic Analysis of Detergent-Resistant Membrane Rafts" (2004). Pharmaceutical Sciences Faculty Publications. 463.