Validation of an Ion Mobility Hydrogen/Deuterium Exchange Mass Spectrometry System

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. Rapid chromatographic separations at 0°C must be utilized to preserve the deuterium label during LC/MS analysis. Unfortunately, fast, low temperature LC separations are not very efficient and can cause spectral overlap for large proteins. The addition of a gas phase, ion mobility separation (IMS) into the HXMS workflow inserts an orthogonal separation that occurs on the millisecond timescale after the LC step without causing any detrimental impact on analysis of deuterium levels. An improved MS system has recently been developed that is capable of higher resolution gas-phase mobility separations. The improved MS platform was combined with a fully automated ultra performance liquid chromatography (UPLC) system developed for HXMS. To evaluate the effect of the new MS platform on deuterium recovery, HXMS experiments were performed using glycogen phosphorylase B as a model protein. Replicate studies of deuterium labeling reactions ranging from 10 seconds to 100 minutes were evaluated. No significant deviation in deuterium levels was observed when using either mobility (HDMSE) or non-mobility (MSE) separations and the standard deviation of deuterium uptake for replicate analyses, for HDMSE and MSE separations were in good agreement with one another. Using the newly developed protocol, an HXMS study of a monoclonal antibody was performed to compare deuterium recovery using a 6 or 12 minute chromatographic separation. Even though chromatographic resolution was reduced with the faster gradient, HDMSE allowed several overlapping peptides to easily be interrogated. In addition, the shorter analysis time improved deuterium recovery for the sample. The application of this additional level of separation will be essential in future studies of very large proteins in which chromatographic efficiency is expected to be suboptimal.