Addressing Limitations in Gas-Phase Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Gas-phase hydrogen/deuterium exchange (gHDX) is a structural mass spectrometry approach that provides information about molecular structure through differences in deuterium incorporation; however, its broader implementation is limited by incomplete understanding of negative-ion exchange behavior, experimental variability, and challenges in measurement comparability. This dissertation addresses these limitations by developing negative-mode gHDX, exchange reporters for standardization, and investigating post-ionization deuterium loss. Evaluation of a chemically diverse panel of anionic compounds demonstrated that exchange behavior is governed by multiple molecular features rather than charge-bearing functional groups alone and enabled identification of reporters spanning slow, intermediate, fast, and broad exchange behaviors. These reporters were used to develop a standardization strategy that successfully corrected changes in gHDX measurements on the Waters Synapt G2-Si; however, different behavior on the Thermo LTQ suggested that an additional component of the exchange environment influenced deuterium incorporation. Investigation of this observation demonstrated that post-ionization deuterium loss occurs across multiple mass spectrometry instruments and in both positive- and negative-ion modes. Targeted humidity control and replacement of vacuum O-rings reduced deuterium loss. The work presented here advances negative-mode gHDX while demonstrating the importance of controlling both the exchange environment and post-ionization preservation of deuterium labeling to improve the reproducibility and reliability of HDX measurements