title: Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins.
abstract: Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of the gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmembrane alpha-helices, the full-length lactose permease from Escherichia coli. Furthermore, ESI-MS is used to titrate reactive thiols with N-ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA. jpw@chem.ucla.edu
authors.forename: J P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: J
authors.initials: J
authors.lastname: le Coutre
authors.forename: J C
authors.initials: JC
authors.lastname: Lee
authors.forename: C K
authors.initials: CK
authors.lastname: Engel
authors.forename: G G
authors.initials: GG
authors.lastname: Privé
authors.forename: K F
authors.initials: KF
authors.lastname: Faull
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease