title: High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins.
abstract: We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography-mass spectrometry (LC-MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2-5 μg of protein. The method is also compatible with our standard LC-MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.
authors.affiliation: Structural Genomics Consortium, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors.forename: Georgina
authors.initials: G
authors.lastname: Berridge
authors.forename: Rod
authors.initials: R
authors.lastname: Chalk
authors.forename: Nazzareno
authors.initials: N
authors.lastname: D'Avanzo
authors.forename: Liang
authors.initials: L
authors.lastname: Dong
authors.forename: Declan
authors.initials: D
authors.lastname: Doyle
authors.forename: Jung-In
authors.initials: JI
authors.lastname: Kim
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Xia
authors.forename: Nicola
authors.initials: N
authors.lastname: Burgess-Brown
authors.forename: Antonio
authors.initials: A
authors.lastname: Deriso
authors.forename: Elisabeth Paula
authors.initials: EP
authors.lastname: Carpenter
authors.forename: Opher
authors.initials: O
authors.lastname: Gileadi
mesh_terms: D002845:Chromatography / Q000295:instrumentation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D000432:Methanol / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D000432:Methanol