Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry.
Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.
Li
Nan
N
Department of Chemistry, University of Alberta, Canada.
Shaw
Andrew R E
AR
Zhang
Nan
N
Mak
Allan
A
Li
Liang
L
D001692:Biological Transport; D002264:Carboxylic Acids / Q000378:metabolism; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D017830:Octoxynol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry
D002264:Carboxylic Acids; D003902:Detergents; D006017:Glycolipids; D008055:Lipids; D008565:Membrane Proteins; D010455:Peptides; D014404:Tubulin; C106354:flotillins; D017830:Octoxynol; D002784:Cholesterol; D014357:Trypsin