Interpreting the Collision Cross Sections of Native-like Protein Ions: Insights from Cation-to-Anion Proton-Transfer Reactions

Abstract

The effects of charge state on structures of native-like cations of serum albumin, streptavidin, avidin, and alcohol dehydrogenase were probed using cation-to-anion proton-transfer reactions (CAPTR), ion mobility, mass spectrometry, and complementary energy-dependent experiments. The CAPTR products all have collision cross section (Ω) values that are within 5.5% of the original precursor cations. The first CAPTR event for each precursor yields products that have smaller Ω values and frequently exhibit the greatest magnitude of change in Ω resulting from a single CAPTR event. To investigate how the structures of the precursors affect the structures of the products, ions were activated as a function of energy prior to CAPTR. In each case, the Ω of the activated precursors increase with increasing energy, but the Ω of the CAPTR products are smaller than the activated precursors. To investigate the stabilities of the CAPTR products, the products were activated immediately prior to ion mobility. These results show that additional structures with smaller or larger Ω can be populated and that the structures and stabilities of these ions depend most strongly on the identity of the protein and the charge state of the product, rather than the charge state of the precursor or the number of CAPTR events. Together, these results indicate that the excess charges initially present on native-like ions have a modest, but sometimes statistically significant, effect on their Ω values. Therefore, potential contributions from charge state should be considered when using experimental Ω values to elucidate structures in solution.

Generation of Charge-Reduced Ions of Membrane Protein Complexes for Native Ion Mobility Mass Spectrometry Studies

“…Electron transfer dissociation has been implemented to reduce the charge state of intact protein complexes post nESI as low as a singly charged species [31,32]. Cation-to-anion protein transfer reactions (CAPTR) is another post ionization technique to reduce the charge state of proteins [33][34][35]. For example, application of CAPTR has revealed that collision cross section of charge-reduced ions generated under denaturing conditions are significantly larger than those generated under native conditions [36].…”

Top-Down Analysis of Proteins in Low Charge States

“…CCSs of proteins in the gas-phase are traditionally measured by ion mobility (IM) methods [40]. Several studies have explored the effect of ESI solvent or PTR on the CCSs of protein ions [41][42][43][44], demonstrating that conformations varied as a function of solution conditions and that charge reduction via PTR did not lead to significant structural collapse. Although IM mass spectrometry is a robust method for measuring CCS of protein ions, estimation of CCS values using an Orbitrap analyzer allows interrogation of the ions on the same platform as used for the HCD and UVPD experiments reported in this study.…”

Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process

“…The opposite is also possible: if Coulomb repulsion is not high enough, non-native non-covalent contacts (not pre-existing in solution) can form in the gas phase and the resulting conformation will be compact. Such gas-phase compaction at low charge states was observed in partially re-neutralized unfolded proteins [28,29], in antibodies [30,31], and in nucleic acid duplexes [32]. A fundamental question in electrospray is thus also what drives a large molecule to adopt a given charge state.…”