Comparison of Partially\nDenatured Cytochrome <i>c</i> Structural Ensembles in Solution\nand Gas Phases Using\nCross-Linking Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) can\nretain intact\nprotein structures, but details about partially folded and unfolded\nprotein structures during and after introduction to the gas phase\nare elusive. Here we use ESI-MS with chemical cross-linkers to compare\ndenatured cytochrome <i>c</i> structures in both solution\nand gas phases. Solution phase cross-linking prior to ESI captures\nsolution phase structures, while gas phase cross-linking through ion/ion\nreactions in the trap cell captures gas phase structures. Comparing\nthe ECD fragmentation of the cross-linked products under both conditions\nshows very similar cross-linker identifications, alluding to no major\nstructural dissimilarities between solution and gas structures. Molecular\nmodeling of the denatured protein using the identified cross-linked\nsites as distant restraints allows for visualization of the denatured\nstructures to pinpoint where unfolding begins. Our data suggest that\ncytochrome <i>c</i> likely begins to unfold due to interior\nhydrophobic expansion, followed by α helical unfolding. This\nlocalization of structural changes is more specific than using CCS\nmeasurements alone.