Direct and Accurate Amide-level Hydrogen/deuterium Exchange Measurements

This grant supports the development of residue-specific resolution Hydrogen/Deuterium exchange with mass spectrometry (HDX-MS), a widespread analytical tool for characterizing proteins. One ongoing limitation with HDX-MS has been that it is inherently a low-resolution technique, often leading to incomplete characterization of protein structure or binding interactions that are critical to understanding how proteins behave and function in solution. The low resolution of established HDX-MS methods also results in poor inter-lab reproducibility. By adopting the use of recently developed mass spectrometry advances in fragmentation strategies along with software development, this work will greatly enhance the resolution offered by HDX-MS to more comprehensively monitor protein structure and binding interfaces, while also directly alleviating ongoing issues with overall reproducibility. These advances will provide a much-needed bioanalytical tool to all researchers for the development, testing, and evaluation of potential therapeutics at both the discovery and development stages. Both graduate and undergraduate students engage in this research to benefit their training and future careers. A main hurdle to HDX-MS studies is the resolution limit imposed by analysis on a peptide-level. Conventional fragmentation approaches result in a high degree of hydrogen migration prior to fragmentation, essentially delocalizing critical information about the exchange profile of each residue within a protein or peptide. Recently emerging alternative fragmentation techniques that utilize electron transfer dissociation and ultraviolet photodissociation are being incorporated into HDX-MS workflows to achieve direct measurement of exchange at the single-residue level. Software developments will streamline both the acquisition steps and the data analysis stages to accomplish amide-resolution HDX-MS without significant decreases in throughput. This project engages in collaborations with both academic labs and industry partners to rigorously evaluate, optimize, and validate the new methods to disseminate effective tools to the greater biochemistry and bioanalytical research communities. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.