title: Proteomic analysis of detergent-resistant membrane rafts.
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.
authors.affiliation: Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, National Cancer Institute, MD, USA.
authors.forename: Josip
authors.initials: J
authors.lastname: Blonder
authors.forename: Martha L
authors.initials: ML
authors.lastname: Hale
authors.forename: David A
authors.initials: DA
authors.lastname: Lucas
authors.forename: Carl F
authors.initials: CF
authors.lastname: Schaefer
authors.forename: Li-Rong
authors.initials: LR
authors.lastname: Yu
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.forename: Haleem J
authors.initials: HJ
authors.lastname: Issaq
authors.forename: Bradley G
authors.initials: BG
authors.lastname: Stiles
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003902:Detergents / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D008563:Membrane Lipids / Q000032:analysis*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000737:chemistry; D014709:Vero Cells
chemical_list: D003902:Detergents; D008563:Membrane Lipids; D008565:Membrane Proteins; D017830:Octoxynol