Effects of charge on protein ion structure: Lessons from cation‐to‐anion, proton‐transfer reactions

Abstract

Collision cross‐section values, which can be determined using ion mobility experiments, are sensitive to the structures of protein ions and useful for applications to structural biology and biophysics. Protein ions with different charge states can exhibit very different collision cross‐section values, but a comprehensive understanding of this relationship remains elusive. Here, we review cation‐to‐anion, proton‐transfer reactions (CAPTR), a method for generating a series of charge‐reduced protein cations by reacting quadrupole‐selected cations with even‐electron monoanions. The resulting CAPTR products are analyzed using a combination of ion mobility, mass spectrometry, and collisional activation. We compare CAPTR to other charge‐manipulation strategies and review the results of various CAPTR‐based experiments, exploring their contribution to a deeper understanding of the relationship between protein ion structure and charge state.

Quantitatively Differentiating Antibodies Using Charge-State Manipulation, Collisional Activation, and Ion Mobility-Mass Spectrometry

“…The 155.6 kDa species is predominant in this spectrum, consistent with the other spectra of this sample (Figures S5 and S9) and that form being predominant in solution. No evidence for a 3.3 kDa fragment ion was observed, consistent with the presence of 155.6 kDa species in solution and the absence of fragmentation in CAPTR experiments . These two species represent different antibody proteoforms in the original sample and may differ in sequence, glycosylation, and other properties.…”

“…The charge states of gas-phase protein ions depend on the size and structure of the protein, the properties of the solvent and other solutes, the ionization mechanism, and other factors. − Charge-state manipulation of protein ions can help probe the effects of charge on the properties of those ions . Several charge-reduction methods have been implemented with MS, − and with IM, it is also possible to probe structural changes in relation to charge state.…”

“…For example, Ω distributions of 8+ ubiquitin appeared to be independent of the applied activation voltage, but Ω distributions of 6+ ions generated by CAPTR of the activated 8+ precursors depended on the extent of activation; these differences provided indirect evidence for unresolved, energy-dependent structural changes in the precursor . Additional results from CAPTR-based experiments and comparisons with results from other charge-reduction methods have been reviewed recently . Overall, these results support the ideas that solution conditions and disulfide bonding impact gas-phase protein ion structure and suggest that charge should be considered when IM-MS is used to study biomolecular structure.…”

Tracking Isomerizations of High-Energy Adenine Cation Radicals by UV–Vis Action Spectroscopy and Cyclic Ion Mobility Mass Spectrometry

“…This includes a new approach to c-IMS whereby we use near-m/z matching of diverse ion precursors for the internal calibration of arrival times of transient species generated by CID. While ion mobility measurements of CCS have become widespread, 53 to our knowledge there have been only a few studies that addressed cation radicals. 54,55 Ion mobility in combination with photodissociation and photoisomerization has been pioneered by Bieske and co-workers to resolve and characterize ion tautomers.…”

“…This includes a new approach to c-IMS whereby we use near- m / z matching of diverse ion precursors for the internal calibration of arrival times of transient species generated by CID. While ion mobility measurements of CCS have become widespread, to our knowledge there have been only a few studies that addressed cation radicals. , Ion mobility in combination with photodissociation and photoisomerization has been pioneered by Bieske and co-workers to resolve and characterize ion tautomers. , We combine the experimental results from UV–vis action spectroscopy and c-IMS with theoretical calculations of ion structures, vibronic absorption spectra, collision cross sections, and isomerization energetics and kinetics to assign structures to adenine cation radicals and track their isomerization in the gas phase.…”