Enhanced resolution of glycosylphosphatidylinositol-anchored and transmembrane proteins from the lipid-rich myelin membrane by two-dimensional gel electrophoresis.
Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2-DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins has slowed the identification of proteins from membrane-rich biological samples. Myelin is an unusually lipid-rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2-DE for the separation of myelin proteins. We have identified two detergents, the nonionic n-dodecyl beta-D-maltoside and the zwitterionic amidosulfobetaine ASB-14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI-anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2-DE. We conclude that these detergents are effective tools for the 2-DE analysis of myelin, and that they may be more generally useful for the analysis of membrane-rich biological samples.
Taylor
Christopher M
CM
Department of Neuroscience, MC 3401, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401, USA. taylor@neuron.uchc.edu
Pfeiffer
Steven E
SE
D000096:Acetone / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002793:Cholic Acids / Q000737:chemistry; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D009186:Myelin Sheath / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D016622:Silver Staining; D013890:Thiourea / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D002793:Cholic Acids; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008055:Lipids; D011506:Proteins; D000096:Acetone; D000431:Ethanol; D014508:Urea; D004492:Edetic Acid; D013890:Thiourea; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D004229:Dithiothreitol