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.

氢氘交换-质谱法（Hydrogen/deuterium exchange-mass spectrometry, HDX-MS）已成为探究蛋白质动态特性的强大工具。作为一种自下而上技术，HDX-MS可提供肽级分辨率的信息，实现动态变化的结构定位。因此，HDX-MS的数据质量在很大程度上取决于氘代后被鉴定和监测的肽段数量。将离子迁移（ion mobility, IM）整合至HDX-MS工作流程中，可通过提供气相中肽离子分离的正交模式提升数据质量，这对于整合膜蛋白（integral membrane proteins, IMPs）等挑战性研究对象尤为关键——这类对象在HDX-MS分析中常面临序列覆盖度低和/或肽段冗余的问题。

随着HDX-MS所研究样本的复杂度不断提升，例如膜模拟重构及体内整合膜蛋白样本，对具备更高分辨能力的仪器的需求应运而生。近期，Giles等人开发了循环离子迁移（cyclic ion mobility, cIM）技术，这是一种采用跑道型几何结构的离子迁移装置，可实现可扩展的多次通过离子迁移分离。

我们借助新近商业化的SELECT SERIES™ Cyclic™ 离子迁移谱仪，采用单次通过与多次通过的cIM流程，对4种去垢剂增溶的整合膜蛋白样品开展HDX-MS分析，并报道了该仪器的增强性能。此外，我们开发了一种新型处理策略，可更好地处理多次通过cIM数据。

值得注意的是，单次通过与多次通过的cIM流程可产生独特的肽段群体，二者结合后的肽段产出量较上一代SYNAPT G2-Si仪器高出31%至222%。据此，我们提出一种集成循环离子迁移的新型HDX-MS工作流程，其有望实现对更复杂系统的高精度、快速度分析。