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

Gozzo, Theresa A.; Bush, Matthew F.

doi:10.1002/mas.21847

Cited by 13 publications

(18 citation statements)

References 111 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Gozzo,

Bush

2023

Anal. Chem.

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Antibody-based therapeutics continue to expand both in the number of products and in their use in patients. These heterogeneous proteins challenge traditional drug characterization strategies, but ion mobility (IM) and mass spectrometry (MS) approaches have eased the challenge of higher-order structural characterization. Energy-dependent IM-MS, e.g., collision-induced unfolding (CIU), has been demonstrated to be sensitive to subtle differences in structure. In this study, we combine a chargereduction method, cation-to-anion proton-transfer reactions (CAPTR), with energy-dependent IM-MS and varied solution conditions to probe their combined effects on the gas-phase structures of IgG1κ and IgG4κ from human myeloma. CAPTR paired with MS-only analysis improves the confidence of chargestate assignments and the resolution of the interfering protein species. Collision cross-section distributions were determined for each of the charge-reduced products. Similarity scoring was used to quantitatively compare distributions determined from matched experiments analyzing samples of the two antibodies. Relative to workflows using energy-dependent IM-MS without charge-state manipulation, combining CAPTR and energy-dependent IM-MS enhanced the differentiation of these antibodies. Combined, these results indicate that CAPTR can benefit many aspects of antibody characterization and differentiation.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Gozzo,

Bush

2023

Anal. Chem.

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Section: ■ Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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We report experimental and computational studies of protonated adenine C-8 σ-radicals that are presumed yet elusive reactive intermediates of oxidative damage to nucleic acids. The radicals were generated in the gas phase by the collision-induced dissociation of C-8−Br and C-8−I bonds in protonated 8-bromoand 8-iodoadenine as well as by 8-bromo-and 8-iodo-9methyladenine. Protonation by electrospray of 8-bromo-and 8iodoadenine was shown by cyclic-ion mobility mass spectrometry (c-IMS) to form the N-1-H, N-9-H and N-3-H, N-7-H protomers in 85:15 and 81:19 ratios, respectively, in accordance with the equilibrium populations of these protomers in water-solvated ions that were calculated by density functional theory (DFT). Protonation of 8-halogenated 9-methyladenines yielded single N-1-H protomers, which was consistent with their thermodynamic stability. The radicals produced from the 8-bromo and 8-iodo adenine cations were characterized by UV−vis photodissociation action spectroscopy (UVPD) and c-IMS. UVPD revealed the formation of C-8 σ-radicals along with N-3-H, N-7-H-adenine πradicals that arose as secondary products by hydrogen atom migrations. The isomers were identified by matching their action spectra against the calculated vibronic absorption spectra. Deuterium isotope effects were found to slow the isomerization and increase the population of C-8 σ-radicals. The adenine cation radicals were separated by c-IMS and identified by their collision cross sections, which were measured relative to the canonical N-9-H adenine cation radical that was cogenerated in situ as an internal standard. Ab initio CCSD(T)/CBS calculations of isomer energies showed that the adenine C-8 σ-radicals were local energy minima with relative energies at 76−79 kJ mol −1 above that of the canonical adenine cation radical. Rice−Ramsperger−Kassel−Marcus calculations of unimolecular rate constants for hydrogen and deuterium migrations resulting in exergonic isomerizations showed kinetic shifts of 10−17 kJ mol −1 , stabilizing the C-8 σ-radicals. C-8 σ-radicals derived from N-1-protonated 9-methyladenine were also thermodynamically unstable and readily isomerized upon formation.

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Perspective: The complex relationship between charge, mobility, and gas‐phase protein structure

Webb

2024

J Mass Spectrom

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Ion mobility spectrometry coupled to mass spectrometry (IMS/MS) is a widely used tool for biomolecular separations and structural elucidation. The application of IMS/MS has resulted in exciting developments in structural proteomics and genomics. This perspective gives a brief background of the field, addresses some of the important issues in making structural measurements, and introduces complementary techniques.

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

Cited by 13 publications

References 111 publications

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

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

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

Perspective: The complex relationship between charge, mobility, and gas‐phase protein structure

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