title: Systematic analysis of the use of amphipathic polymers for studies of outer membrane proteins using mass spectrometry.
abstract: Membrane proteins (MPs) are essential for numerous important biological processes. Recently, mass spectrometry (MS), coupled with an array of related techniques, has been used to probe the structural properties of MPs and their complexes. Typically, detergent micelles have been employed for delivering MPs into the gas-phase, but these complexes have intrinsic properties that can limit the utility of structural studies of MPs using MS methods. Amphipols (APols) have advantages over detergent micelles and have been shown to be capable of delivering native MPs into the gas-phase. Comparing six different APols which vary in mass and charge, and the detergentn-dodecyl-β-d-maltopyranoside, we aimed to determine which APols are most efficient for delivery of native outer membrane proteins (OMPs) into the gas-phase. We show that maintaining the solution-phase folding and global structures of three different OMPs (PagP, OmpT and tOmpA) are independent of the APol used, but differences in OMP activity can result from the different APol:OMP complexes. ESI-IMS-MS analysis of OMP:APol complexes shows that the A8-35 APol is most proficient at liberating all three OMPs into the gas-phase, without altering their gas-phase conformations.
authors: Astbury Centre for Structural Molecular Biology, School of Molecular & Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors: Thomas G
authors: TG
authors: Watkinson
authors: Astbury Centre for Structural Molecular Biology, School of Molecular & Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors: Antonio N
authors: AN
authors: Calabrese
authors: Laboratoire de Physico-Chimie Moléculaire des Protéines Membranaires, UMR 7099, Institut de Biologie Physico-Chimique (FRC 550), Centre National de la Recherche Scientifique/Université Paris-7, 13, rue Pierre-et-Marie-Curie, 75005 Paris, France.
authors: Fabrice
authors: F
authors: Giusti
authors: Laboratoire de Physico-Chimie Moléculaire des Protéines Membranaires, UMR 7099, Institut de Biologie Physico-Chimique (FRC 550), Centre National de la Recherche Scientifique/Université Paris-7, 13, rue Pierre-et-Marie-Curie, 75005 Paris, France.
authors: Manuela
authors: M
authors: Zoonens
authors: Astbury Centre for Structural Molecular Biology, School of Molecular & Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors: Sheena E
authors: SE
authors: Radford
authors: Astbury Centre for Structural Molecular Biology, School of Molecular & Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors: Alison E
authors: AE
authors: Ashcroft
keywords: Amphipols; Electrospray ionisation–mass spectrometry; Ion mobility spectrometry–mass spectrometry; Outer membrane proteins; Protein conformation