Covalent Cross-Linking as an Enabler for Structural Mass Spectrometry

The number of studies referring to the structural elucidation of intact biomolecular systems using mass spectrometry techniques has gradually increased in the post-2000s literature topics. As part of native mass spectrometry, this domain capitalizes on the kinetic trapping of physiological folds in view of probing solution-like conformational properties of isolated molecules or complexes after their electrospray transfer to the gas phase. Despite its efficiency for a wide array of analytes, this approach is expected to be pushed to its limits when considering highly dynamic systems or when dealing with nonideal operating conditions. To circumvent these limitations, we challenge the adequacy of an original strategy based on cross-linkers to improve the gas-phase stability of isolated proteins and ensure the preservation of folded conformations when measuring with strong transmission voltages, by spraying from denaturing solvents, or trapping for extended periods of time. Tested on cytochrome c, myoglobin, and beta-lactoglobulin cross-linked using BS3, we validated the process as structurally nonintrusive in solution using far-ultraviolet circular dichroism and unraveled the preservation of folded conformations showing better resilience to denaturation on cross-linked species using ion mobility. The resulting collision cross sections were found in agreement with the native fold, and a preservation of the proteins' secondary and tertiary structures was evidenced using molecular dynamics simulations. Our results provide new insights concerning the fate of electro-sprayed cross-linked conformers in the gas phase, while constituting promising evidence for the validation of this technique as part of future structural mass spectrometry workflows.