Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes

Abstract

Membrane protein characterization is consistently hampered by challenges with expression, purification, and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and Q-ToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ), under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation, is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation. Graphical Abstract ᅟ.

Native-MS Analysis of Monoclonal Antibody Conjugates by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

“…However, over the past 5 years more native gas-phase protein and high mass applications have been described by FT-ICR where the applications have ranged from mAbs 27 , to membrane proteins 49,50 , nanodiscs 49,51,52 and native protein top-down analyses 53,54 . The native-MS data presented herein further demonstrates the enabling capabilities of an unmodified FT-ICR platform for native high mass (and high m/z) analysis, producing mAb conjugate data equivalent in quality to that previously only thought to be achievable using oa-ToF analysers [55][56][57][58][59] and more recently the Orbitrap 19,23,26,60 .…”

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…36,41 By using appropriate sample preparation, ionisation conditions, and instrumentation, many challenging analytes such as membrane proteins, 18,42 intrinsically disordered proteins, 43 highly dynamic or heterogeneous complexes, 19 or very large systems such as intact virus capsids 44 can all be investigated using native MS, as well as their associated proteoforms. 45 Many of these are highly challenging to study by other analytical techniques such as NMR or XRD, and therefore the insights from MS can be vital to understanding these proteins and complexes. It is worth noting here that, while lower-resolution, MS-based methods are not necessarily less native than conventional methodsthe crystalline state is far removed indeed from the native protein environment.…”

Surface-Induced Dissociation: An Effective Method for Characterization of Protein Quaternary Structure

“…FT-ICR instruments have more recently also been used in native mass spectrometry and structural biology applications, 162–164 including structural characterization, 95,165,166 top-down dissociation, 67,92 ligand binding, 167,168 and membrane protein studies. 169,170 In 2017, Yan et. al.…”