Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins.
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.
Whitelegge
J P
JP
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA. jpw@chem.ucla.edu
le Coutre
J
J
Lee
J C
JC
Engel
C K
CK
Privé
G G
GG
Faull
K F
KF
Kaback
H R
HR
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
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