| Hydrogen/deuterium exchange (H/DX), in combination with mass spectrometry (MS), has emerged as a powerful and convenient biophysical tool to map protein structure, dynamics, and ligation. While great strides have been made since its inception, continued methodological advancements are required to enable the analysis of large protein complexes (>200 kDa) and low abundant components in these complexes. Such analyses will undoubtedly present analytical challenges related to sensitivity, spectral interference, and speed of analysis. To address these issues, a series of incremental approaches for conducting peptide-based H/DX-MS measurements were developed and applied to an array of protein mixtures prepared under near-physiological conditions. The first involved controlling the expansion of the peptide isotopic envelope through an alternative approach to labelling and data extraction. It was demonstrated that precise and sensitive measurements to a structural perturbation can be preserved through a significant reduction in the %D2O applied in the labelling reaction. Further, a minimum of two peaks of a peptide peak distribution, as opposed to the entire envelope, can suffice in monitoring changes in protein structure and dynamics. Using these findings, the utility of the tandem MS domain, operated in the data-dependent acquisition (DDA) and multiple reaction monitoring (MRM) modes, was then explored for quantitating deuteration levels and improving the analytical figures of merit; thus, providing an additional dimension for analysis. In the DDA mode, despite pervasive intrapeptide H/D scrambling in the gas-phase upon collision-induced dissociation, differential deuteration levels for product ions (with the exception of neutral loss products) were found to be in general agreement with values obtained from their corresponding precursors. In the MRM mode, an approach for targeting sequence was developed through mass shift perturbation detection of select peptides. Using this approach, shift measurement precision was demonstrated to match that of conventional high resolution determinations, exceed the dynamic range, and simplify data analysis. Lastly, to generate an accurate and reliable structural model of a protein complex, the H/DX-MS technique was partnered with computational modeling. Here, theoretical H/DX-MS data on a heteromeric protein complex was used to drive molecular docking simulations, in a manner that is applicable to real data. |
