Abstract
Determining structures of multiply charged protein ions in mass spectrometry is critical for understanding the relationship between condensed-phase protein structures found in biological environments and gas-phase structures produced during electrospray ionization (ESI). The determination of collision cross-sections (CCSs) via ion mobility-mass spectrometry (IM-MS) has been used to study conformational changes as a function of protein charge state; however, this approach provides limited structural information on the overall topology of analytes. Recently developed gas-phase cross-linking mass spectrometry (XL-MS) performed via ion/ion reactions can probe local tertiary structures, providing an additional dimension to MS-based gas-phase structural biology studies. Herein, we demonstrate the conformational differentiation of alcohol-denatured, ESI-generated ubiquitin ions with varying charges (6+ through 11+) using gas-phase XL-MS. Gas-phase N-hydroxysulfosuccinimide (NHS) ester chemistry is performed using sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linkers to covalently modify neutral and charged basic amino acid residues. This method provides sufficient sensitivity to detect subtle conformational changes in local tertiary structures specific to each charge state. These results reveal that 6+ through 8+ charge states of ubiquitin adopt folded conformations, while the 9+ through 11+ charge states exhibit unfolded structures. This trend is consistent with the bimodal charge state distribution observed in the ESI mass spectrum of alcohol-denatured ubiquitin. Overall, this gas-phase XL-MS method enables the examination of conformational changes in gas-phase proteins of varying charge states at the local tertiary structural motif level, providing a novel gas-phase structural biology approach that complements existing MS-based methods.