Local hydrogen deuterium exchange mass spectrometry: From pressurized online digestion to pepsin proteolysis

Higher-order structure of a protein is important for proper biological function. Current analytical and structural methods provide comprehensive understanding of the chemical, physical, and biological characteristics of biologics, yet detecting a change in dynamics and conformation can be a challenging task. Hydrogen deuterium exchange mass spectrometry (HDX MS) can be used to probe conformational changes of proteins. It is particularly useful to detect subtle changes in conformation at the peptide level. It is critical to generate many different kinds of overlapping peptides that are reproducible, so they can be used to enhance sequence coverage and spatial resolution.;The primary goal of this study focused on how local HDX MS can be improved and refined using peptides produced during pepsin digestion. First, a unique way to pressurize during online pepsin digestion was developed to be able to run the HDX experiments and digestion efficiency was investigated under pressure. Pepsin was immobilized on bridged ethylene hybrid (BEH) particles and digestion performance was evaluated in a completely online format, with the specific intent of using the particles for HDX MS experiments. Because BEH particles are mechanically strong, they could withstand prolonged, continuous high-pressure at 10,000 psi. Digestion efficiency and the reproducibility of peptic peptides at 10,000 psi were compared with 1,000 psi digestions. Columns made with the BEH pepsin particles were evaluated for robustness and deuterium back-exchange and were compared to other immobilized pepsin particles using phosphorylase b, G-CSF, and other proteins. This study described how these BEH particles can be incorporated in existing HDX MS workflows to provide more peptide coverage in experiments where fast, efficient, and reproducible online pepsin digestion is desired.;The reproducibility and specificity of online pepsin digestions were investigated qualitatively and quantitatively. The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in HDX MS experiments for digestion under hydrogen exchange quench conditions. Even though the specificity of pepsin has been studied previously, further investigation has been completed to understand the reproducibility and specificity of digestion. These data showed that the number of reproducible peptides in pepsin digestions becomes constant even as the number of pepsin digestions is increased.;Finally, a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) was introduced for local HDX MS analysis and its digestion efficiency and specificity were compared to porcine pepsin and aspergillopepsin. Unique cleavage specificity was determined for rice field eel pepsin. Generating different peptides produced by the various proteases showed how protein sequence coverage and the spatial resolution of HDX MS data can be enhanced.