Gas-Phase Protein Salt Bridge Stabilities from Collisional Activation and Electron Transfer Dissociation.

The gas phase structures of several proteins have been studied by electron transfer dissociation (ETD) with and without prior collisional heating after electrospraying these proteins from native-like solutions into a quadrupole ion trap mass spectrometer. Without prior collisional heating, we find that ETD fragmentation is mostly limited to regions of the protein that are not spanned by the salt bridges known to form in solution. When protein ions are collisionally heated before ETD, new product ions are observed, and in almost all cases, these new ions arise from protein regions that are spanned by the salt bridges. Together these results confirm the existence of salt bridges in protein ions and demonstrate that a sufficient amount energy is required to disrupt these salt bridges in the gas phase. More interestingly, we also show that different salt bridges require different collisional activation voltages to be disrupted, suggesting that they have variable stabilities in the gas phase. These stabilities appear to be influenced by the gas-phase basicities of the involved residues and the presence of nearby charged residues. We also find that higher collisional activation voltages are needed to enable the formation of new product from sites spanned by multiple salt bridges.