Mistarz Ulrik H, Brown Jeffery M, Haselmann Kim F, Rand Kasper D
Department of Pharmacy, University of Copenhagen , Universitetsparken 2, 2100 Copenhagen, Denmark.
Anal Chem. 2014 Dec 2;86(23):11868-76. doi: 10.1021/ac5035456. Epub 2014 Nov 20.
Gas-phase hydrogen/deuterium exchange (HDX) is a fast and sensitive, yet unharnessed analytical approach for providing information on the structural properties of biomolecules, in a complementary manner to mass analysis. Here, we describe a simple setup for ND3-mediated millisecond gas-phase HDX inside a mass spectrometer immediately after ESI (gas-phase HDX-MS) and show utility for studying the primary and higher-order structure of peptides and proteins. HDX was achieved by passing N2-gas through a container filled with aqueous deuterated ammonia reagent (ND3/D2O) and admitting the saturated gas immediately upstream or downstream of the primary skimmer cone. The approach was implemented on three commercially available mass spectrometers and required no or minor fully reversible reconfiguration of gas-inlets of the ion source. Results from gas-phase HDX-MS of peptides using the aqueous ND3/D2O as HDX reagent indicate that labeling is facilitated exclusively through gaseous ND3, yielding similar results to the infusion of purified ND3-gas, while circumventing the complications associated with the use of hazardous purified gases. Comparison of the solution-phase- and gas-phase deuterium uptake of Leu-Enkephalin and Glu-Fibrinopeptide B, confirmed that this gas-phase HDX-MS approach allows for labeling of sites (heteroatom-bound non-amide hydrogens located on side-chains, N-terminus and C-terminus) not accessed by classical solution-phase HDX-MS. The simple setup is compatible with liquid chromatography and a chip-based automated nanoESI interface, allowing for online gas-phase HDX-MS analysis of peptides and proteins separated on a liquid chromatographic time scale at increased throughput. Furthermore, online gas-phase HDX-MS could be performed in tandem with ion mobility separation or electron transfer dissociation, thus enabling multiple orthogonal analyses of the structural properties of peptides and proteins in a single automated LC-MS workflow.
气相氢/氘交换（HDX）是一种快速且灵敏但尚未得到充分利用的分析方法，可用于以与质谱分析互补的方式提供生物分子结构特性的信息。在此，我们描述了一种在电喷雾电离（ESI）后立即在质谱仪内部进行由ND₃介导的毫秒级气相HDX的简单装置（气相HDX-MS），并展示了其在研究肽和蛋白质的一级及高级结构方面的实用性。通过使氮气通过装有氘代氨水溶液试剂（ND₃/D₂O）的容器，并在一级撇气锥的上游或下游立即引入饱和气体来实现HDX。该方法在三种商用质谱仪上得以实施，且无需或只需对离子源的气体入口进行少量完全可逆的重新配置。使用ND₃/D₂O水溶液作为HDX试剂对肽进行气相HDX-MS的结果表明，标记仅通过气态ND₃来促进，产生的结果与注入纯化的ND₃气体相似，同时避免了与使用危险纯化气体相关的复杂性。亮氨酸脑啡肽和谷氨酸纤维蛋白肽B的溶液相和气相氘摄取的比较证实，这种气相HDX-MS方法允许标记经典溶液相HDX-MS无法触及的位点（位于侧链、N端和C端的杂原子结合的非酰胺氢）。该简单装置与液相色谱和基于芯片的自动纳升电喷雾电离接口兼容，能够在液相色谱时间尺度上以更高的通量对分离的肽和蛋白质进行在线气相HDX-MS分析。此外，在线气相HDX-MS可以与离子淌度分离或电子转移解离串联进行，从而能够在单个自动化液相色谱-质谱工作流程中对肽和蛋白质的结构特性进行多次正交分析。
