Comparison of Partially Denatured Cytochrome <i>c</i> 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 <i>c</i> 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 <i>c</i> 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.