Development and Validation of a UPLC Hydrogen/Deuterium Exchange Mass Spectrometry System

RP-81 Hydrogen/deuterium exchange mass spectrometry (HXMS) has proven to be a useful analytical method for the study of protein dynamics and changes to protein conformation. In order to maximize deuterium signal recovery during LC/MS analysis, rapid chromatographic separations at 0°C must be utilized. Recently, an ultraperformance liquid chromatograpy (UPLC) system has been developed capable of high resolution separations at 0°C. In this study, we describe a comprehensive HXMS workflow which is capable of chromatographic separations ranging from 300 μm to 1.0 mm i.d columns. Online pepsin digestion was utilized for rapid, reproducible protein digestion. Peptides were fragmented by a simple scheme of alternating scans with low and high collision energy (MSE). To validate the system hardware performance, a standard protocol has been developed to evaluate all aspects of the workflow. MSE analyses show high confidence peptide identification, up to 100% linear sequence coverage, and reproducible peptic peptide peak area (less than 10 % RSD) in large scale replicate analyses of phosphorylase b. Results using an automated robotics platform utilized for sample preparation will also be discussed.