An Integrated System for Studying Protein Structure and Dynamics by Hydrogen Exchange Mass Spectrometry

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This proposal is for a nanoACQUITY/HDX system that is required for studying protein structure and dynamics by mass spectrometry (MS). The cellular functions of proteins are intimately linked to their structures. However, proteins also have to undergo a multitude of conformational motions to perform their biological tasks. This structure/function/dynamics triad remains poorly understood. Pioneering work by Konermann and others has revealed that hydrogen/deuterium exchange (HDX) MS is a powerful tool for addressing this problem. Protein regions that are highly flexible undergo rapid HDX. Isotope exchange is much slower in rigid segments. The HDX workflow involves digestion and subsequent peptide analysis by liquid chromatography/MS under quench conditions. The deuteration kinetics of individual protein segments can be deduced from the resulting mass profiles. The recent development of a commercial HDX-MS platform has dramatically changed the playing field. Several US labs already purchased this nanoACQUITY/HDX technology, but none of these systems are available in Canada yet. This new platform provides greatly improved reproducibility, digestion efficiency, robustness, and software ease-of-use compared to conventional home-built systems. The proposed equipment will allow a significant expansion of Konermann's research program into the area of transmembrane proteins, many of which represent important drug targets. Specifically, the nanoACQUITY/HDX will allow studies on transmembrane permeation, the mechanism of molecular motors (with Dunn), as well as the structural characterization of bacterial membrane enzymes (with Valvano). Using funds obtained elsewhere, Konermann was able to purchase a state-of-the-art Synapt mass spectrometer that represents one half of the HDX platform. The nanoACQUITY/HDX represents the second half that is still missing (for liquid handling/digestion/chromatography). The current proposal offers superb opportunities for HQP training. Failure to secure funding for this equipment would be a severe setback for the applicants' research programs.