Cyclic ion mobility for hydrogen/deuterium exchange-mass spectrometry applications

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localisation of dynamic changes. Consequently, HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase data quality by providing an orthogonal mode of peptide ion separation in the gas-phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage and/or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multi-pass IM separations. Using 1-pass and multi-pass cIM routines, we use the recently commercialised SELECT SERIES™ Cyclic™ IM spectrometer for HDX-MS analyses of 4 detergent solubilised IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multi-pass cIM data. Interestingly, use of 1-pass and multi-pass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM which has the potential to enable the analysis of more complex systems with greater accuracy and speed.