title: Native MS Analysis of Bacteriorhodopsin and an Empty Nanodisc by Orthogonal Acceleration Time-of-Flight, Orbitrap and Ion Cyclotron Resonance.
abstract: Over the past two decades, orthogonal acceleration time-of-flight has been the de facto analyzer for solution and membrane-soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Three MS instruments are compared, the Q-ToF, Orbitrap, and the FT-ICR, to analyze, under native instrument and buffer conditions, the seven-transmembrane helical protein bacteriorhodopsin-octylglucoside micelle and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode), producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20-0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29-2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on all three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge states. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa), and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.
authors.forename: Iain D G
authors.identifier: 0000-0003-4310-8540
authors.initials: ID
authors.lastname: Campuzano
authors.affiliation: Department of Chemistry and Biochemistry, and Department of Biological Chemistry, University of California-Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.affiliation: Discovery Analytical Sciences, Amgen , South San Francisco, California 94080, United States.
authors.forename: Dhanashri
authors.initials: D
authors.lastname: Bagal
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Lippens
authors.forename: Juraj
authors.initials: J
authors.lastname: Svitel
authors.forename: Robert J M
authors.initials: RJ
authors.lastname: Kurzeja
authors.forename: Han
authors.initials: H
authors.lastname: Xu
authors.affiliation: Discovery Analytical Sciences, Amgen , South San Francisco, California 94080, United States.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Schnier
authors.affiliation: Department of Chemistry and Biochemistry, and Department of Biological Chemistry, University of California-Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry*; D017357:Cyclotrons; D005583:Fourier Analysis; D005960:Glucosides / Q000737:chemistry*; D019617:Halobacterium salinarum / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008823:Micelles; D008958:Models, Molecular; D049329:Nanostructures / Q000737:chemistry; D011487:Protein Conformation; D018717:Purple Membrane / Q000737:chemistry
chemical_list: D005960:Glucosides; D008823:Micelles; C018619:octyl-beta-D-glucoside; D001436:Bacteriorhodopsins