Pharmaceutical Sciences Faculty Publications

Title

Proteomic Analysis of Detergent-Resistant Membrane Rafts

Document Type

Article

Publication Date

5-1-2004

Journal Title

Electrophoresis

ISSN

0173-0835

Volume

25

Issue

9

First Page

1307

Last Page

1318

DOI

10.1002/elps.200405891

PubMed ID

15174053

Abstract

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.

Keywords

Amino acid sequence, cell membrane, chlorocebus aethiops, detergents, gas chromatography-mass spectrometry, membrane, lipids, microdomains, proteins, octoxynol, vero cells

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