New Insights into the Intrinsic Electron-Based Dissociation Behavior of Cytochrome c Oligomers.

Between 2003 and 2017, four reports were published that demonstrated the intrinsic ability of the native iron-containing proteins cytochrome c and ferritin to undergo radical-based backbone fragmentation in the gas phase without the introduction of exogenous electrons. For cytochrome c in particular, this effect has so far only been reported to occur in the ion source, preventing the in-depth study of reactions occurring after gas-phase isolation of specific precursors. Here, we report the first observation of this intrinsic native electron capture dissociation behavior after quadrupole isolation of specific charge states of the cytochrome c dimer and trimer, providing direct experimental support for key aspects of the mechanism proposed 20 years ago. Furthermore, we provide evidence that, in contrast to some earlier proposals, these oligomeric states are formed in bulk solution rather than during the electrospray ionization process and that the observed fragmentation site preferences can be rationalized through the structure and interactions within these native oligomers rather than the monomer. We also show that the observed fragmentation pattern─and indeed, whether or not fragmentation occurs─is highly sensitive to the provenance and history of the protein samples, to the extent that samples can show distinct fragmentation behavior despite behaving identically in ion mobility experiments. This rather underexplored method therefore represents an exquisitely sensitive conformational probe and will hopefully receive more attention from the biomolecular mass spectrometry community in the future.