Native MS Analysis of Bacteriorhodopsin and an Empty Nanodisc by Orthogonal Acceleration Time-of-Flight, Orbitrap and Ion Cyclotron Resonance

Anal Chem. 2016 Dec 20;88(24):12427-12436. doi: 10.1021/acs.analchem.6b03762. Epub 2016 Dec 1.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteriorhodopsins / chemistry*
  • Cyclotrons
  • Fourier Analysis
  • Glucosides / chemistry*
  • Halobacterium salinarum / chemistry
  • Mass Spectrometry / methods*
  • Micelles*
  • Models, Molecular
  • Nanostructures / chemistry
  • Protein Conformation
  • Purple Membrane / chemistry

Substances

  • Glucosides
  • Micelles
  • octyl-beta-D-glucoside
  • Bacteriorhodopsins