Comparison of Partially Denatured Cytochrome Structural Ensembles in Solution and Gas Phases Using Cross-Linking Mass Spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) can retain intact protein structures, but details about partially folded and unfolded protein structures during and after introduction to the gas phase are elusive. Here we use ESI-MS with chemical cross-linkers to compare denatured cytochrome structures in both solution and gas phases. Solution phase cross-linking prior to ESI captures solution phase structures, while gas phase cross-linking through ion/ion reactions in the trap cell captures gas phase structures. Comparing the ECD fragmentation of the cross-linked products under both conditions shows very similar cross-linker identifications, alluding to no major structural dissimilarities between solution and gas structures. Molecular modeling of the denatured protein using the identified cross-linked sites as distant restraints allows for visualization of the denatured structures to pinpoint where unfolding begins. Our data suggest that cytochrome likely begins to unfold due to interior hydrophobic expansion, followed by α helical unfolding. This localization of structural changes is more specific than using CCS measurements alone.