Open AccessToward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins
Author(s) -
Julian P. Whitelegge,
Johannes le Coutre,
J. C. Lee,
C.K. Engel,
Gilbert G. Privé,
Kym F. Faull,
H. Ronald Kaback
Publication year - 1999
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.96.19.10695
Subject(s) - electrospray ionization , chemistry , proteome , membrane protein , lactose permease , transmembrane protein , mass spectrometry , escherichia coli , biochemistry , tandem mass spectrometry , chromatography , membrane , membrane transport protein , gene , receptor
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 α-helices, the full-length lactose permease fromEscherichia coli . Furthermore, ESI-MS is used to titrate reactive thiols withN -ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.