Top-down protein analysis by chemical cross-linking mass spectrometry (XL-MS) has emerged as a useful tool for assessing intramolecular interactions and tertiary structures of proteins via the distance constraints imposed by the cross-linkers. Soft ionization methods, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), are routinely employed to introduce protein ions from the condensed phase into the gas phase for XL-MS. The sites of linker attachments are determined by tandem mass spectrometry (MS2) of the modified proteins following cross-linking. While typically performed in solution, cross-linking reactions can be performed in the gas phase via ion/ion reactions, reducing the sample preparation time. Herein, we demonstrate the conformational differentiation of ubiquitin ions originating from ESI and MALDI of denaturing solution via gas-phase ion/ion XL-MS on a commercial Fourier transform ion cyclotron resonance (FTICR) MS equipped with infrared multiphoton dissociation (IRMPD) for MS2. Ubiquitin is known to exist predominantly in the A-state under denaturing solution conditions. Interestingly, the presence of both charge-state-associated folded (8+ through 6+) and unfolded (8+ through 11+) structures derived from the original A-state solution conformation is indicated via our gas-phase ion/ion XL-MS method using ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) as a cross-linker. Further investigation into the 6+ ubiquitin ion produced via ESI from denaturing solutions reveals structural similarity in the N-terminal beta-hairpin motif relative to the 6+ counterpart produced from native-like solutions (i.e., Lys6 and Lys11 on both conformers are cross-linked by sulfo-EGS). In order to determine the level of conformational similarity between the two versions of 6 + ubiquitin ions, cross-linkers of shorter lengths are currently being employed. Compared to the conformations from ESI-generated 6+ charge states, the gas-phase ion/ion XL-MS of 3+ and 4+ ubiquitin ions generated via MALDI of denaturing solutions reveals more compact conformations with stronger intramolecular noncovalent interactions.

Gas-phase intramolecular cross-linking via ion/ion reactions coupled with IRMPD for structural differentiation of protein conformers

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