Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana 46202, United States.
Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States.
Anal Chem. 2022 Jun 28;94(25):8975-8982. doi: 10.1021/acs.analchem.2c00942. Epub 2022 Jun 16.
In this article, we present an approach for conformationally multiplexed, localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-IM-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND) to be leaked in after / selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. Gas-phase HDX with ECD fragmentation for exchange site localization was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome . Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially folded A-state. IM of deuterated ubiquitin 6 ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5 charge state showed many regions with protection from HDX, and the 11 charge state, ionized from conditions that stabilize the A-state, showed high levels of deuterium incorporation throughout most of the protein sequence. The 7 ions of cytochrome ionized from aqueous conditions showed greater HDX with unstructured regions of the protein relative to interior, structured regions, especially those involved in heme binding. With careful tuning and attention to deuterium scrambling, our approach holds promise for determining region-specific information on a conformer-selected basis for gas-phase protein structures, including localized characterizations of ligand, epitope, and protein-protein binding.
在本文中，我们提出了一种方法，通过离子淌度（IM）促进的气相蛋白质离子的构象多重、局部氢氘交换（HDX），随后进行电子捕获解离（ECD）。先前经过修改的四极杆-IM-飞行时间仪器，可在无需离子捕获的情况下在传输模式下进行 ECD（没有离子陷阱），随后进一步修改以允许在/选择后泄漏重氮氨（ND）。最小化碰撞激活以防止氘交换产生与结构无关的结果。用广泛研究的蛋白质折叠模型泛素和细胞色素 进行了带有 ECD 片段的气相 HDX 以确定交换位点定位。泛素从稳定天然状态的条件和稳定部分折叠 A 状态的条件下被离子化。氘化泛素 6 离子的 IM 允许从更紧凑的构象体中分离出更扩展的构象体。分离的子群体的 ECD 显示，更扩展的（较晚到达的）构象体在观察到的 HDX 量上具有显著的、局部的差异。5 价态显示出许多具有 HDX 保护的区域，而从稳定 A 状态的条件下离子化的 11 价态则显示出在整个蛋白质序列的大部分区域都具有高水平的氘掺入。从水相条件下离子化的细胞色素 7 离子显示出与蛋白质内部结构区域相比，蛋白质无结构区域具有更大的 HDX，特别是那些与血红素结合相关的区域。通过仔细调整和注意氘交换，我们的方法有望确定气相蛋白质结构的构象选择基础上的区域特异性信息，包括配体、表位和蛋白质-蛋白质结合的局部特征。
