title: Hydrophobic peptide mapping of clinically relevant heptathelical membrane proteins by capillary electrophoresis.
abstract: The structural investigation of G protein-coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid-based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrene-coated fused silica capillary. The potential for detection sensitivity to be increased at least 100-fold by use of on-line solid-phase extraction on C18-silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membrane receptors.
authors.affiliation: Center for Basic Research in Digestive Diseases, Rochester, MN 55905, USA.
authors.forename: M
authors.initials: M
authors.lastname: Dong
authors.forename: R P
authors.initials: RP
authors.lastname: Oda
authors.forename: M A
authors.initials: MA
authors.lastname: Strausbauch
authors.forename: P J
authors.initials: PJ
authors.lastname: Wettstein
authors.forename: J P
authors.initials: JP
authors.lastname: Landers
authors.forename: L J
authors.initials: LJ
authors.lastname: Miller
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry*; D003488:Cyanogen Bromide; D019075:Electrophoresis, Capillary / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D012995:Solubility; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D012997:Solvents; D001436:Bacteriorhodopsins; D003488:Cyanogen Bromide