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 (ND₃) to be leaked in after m/z 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 c. 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 c 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.

中文翻译：

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通过传输模式电子捕获解离实现蛋白质离子的多重构象选择性、局部气相氢氘交换

在本文中，我们提出了一种通过离子迁移 (IM) 和电子捕获解离 (ECD) 促进气相蛋白质离子的构象多路复用、局部氢氘交换 (HDX) 的方法。之前修改的四极杆-IM-飞行时间仪器可以在迁移率分离后立即以传输模式（无离子捕获）启用 ECD，进一步修改以允许氘化氨（ND ₃）在m / z选择后泄漏。碰撞激活被最小化以防止氘扰乱给出结构上不相关的结果。使用广泛研究的蛋白质折叠模型泛素和细胞色素证明了具有 ECD 片段化用于交换位点定位的气相 HDX丙。泛素从稳定天然状态的条件和稳定部分折叠的 A 状态的条件中电离。氘化泛素 6 ⁺离子的 IM 允许更紧凑的构象异构体与更多扩展的构象异构体分离。分离的亚群的 ECD 显示，更扩展（较晚到达）的构象异构体在观察到的 HDX 量方面具有显着的局部差异。5 ⁺电荷状态显示许多区域受到 HDX 的保护，而 11 ⁺电荷状态从稳定 A 状态的条件下电离，在大部分蛋白质序列中显示出高水平的氘掺入。细胞色素c的 7 ^+离子从水性条件下电离的 HDX 与蛋白质的非结构化区域相比显示出更大的 HDX，相对于内部的结构化区域，尤其是那些参与血红素结合的区域。通过仔细调整和关注氘加扰，我们的方法有望在气相蛋白质结构的构象异构体选择基础上确定区域特异性信息，包括配体、表位和蛋白质-蛋白质结合的局部表征。