Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins

Genes encoding membrane proteins comprise a substantial proportion of genom es sequenced to date, but ability to perform structural studies on this por tion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of th e gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmem brane alpha-helices, the full-length lactose permease from Escherichia coli . Furthermore, ESI-MS is used to titrate reactive thiols with N-ethylmaleim ide. Treatment of the native protein solubilized in detergent micelles reve als only two reactive thiols, and both are protected by a substrate analog.