Protein Structural Characterization by Hydrogen/Deuterium Exchange Mass Spectrometry with Top-down Electron Capture Dissociation

This study tested the feasibility of observing HID exchange of intact protein by top-down electron capture dissociation (ECD) mass spectrometry for the investigation of protein structure. Ubiquitin is selected as a model system. Local structural information was obtained from the deuteration levels of c and z. ions generated from ECD. Our results showed that alpha-helix region has the lowest deuteration level and the C-terminal fraction containing a highly mobile tail has the highest deuteration level, which correlates well with previous X-Ray and HDX/NMR. analyses. We studied site-specific H/D exchange kinetics by monitoring HID exchange rate of several structural motives of ubiquitin. Two hydrogen bonded beta-strands showed similar HDX rates. However, the outer beta-strand always has higher deuteration level than the inner beta-strand. The HDX rate of the turn structure (residues 8-11) is lower than that of beta-strands (residues 1-7 and residues 12-17) it connects. Although isotopic distribution gets broader after H/D exchange which results in a limited number of backbone cleavage sites detected, our results demonstrate that this method can provide valuable detailed structural information of proteins. This approach should also be suitable for the structural investigation of other unknown proteins, protein conformational changes, as well as protein-protein interactions and dynamics.