Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution.
Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution.
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DOI：
10.1021/acs.analchem.2c05310
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发表时间：
2023-03-21
影响因子：
7.4
通讯作者：
Phillips, Jonathan J.
中科院分区：
文献类型：
--
作者：
Kish, Monika;Smith, Victoria;Lethbridge, Natasha;Cole, Lindsay;Bond, Nicholas. J.;Phillips, Jonathan J.
酰胺氢/氘交换质谱（HDX-MS）是分析蛋白质在溶液中构象动力学的有力工具。目前的传统方法的测量极限从几秒开始，并且完全依赖于手动移液或液体处理机器人的速度。多肽的弱保护区，如短肽，暴露环和本质上无序的蛋白质交换毫秒时间尺度。典型的HDX方法往往不能解决这些情况下的结构动力学和稳定性。许多学术实验室已经证明了在亚秒级范围内获取HDX-MS数据的相当大的实用性。在这里，我们描述了一个完全自动化的HDX-MS装置的发展，以解决酰胺交换毫秒的时间尺度。与传统系统一样，该仪器拥有自动进样功能，可通过软件选择标记时间、在线流动混合和淬灭，同时与液相色谱-MS系统完全集成，可用于现有的标准“自下而上”工作流程。HDX-MS对几种肽的快速交换动力学证明了该系统实现的重复性、再现性、反向交换和混合动力学。相比之下，273个肽的肽覆盖率为96.4%，支持系统与标准机器人的等效性。此外，50 ms-300 s的时间窗允许观察到许多酰胺基团的完全动力学转变;对于可能高度动态和溶剂暴露的区域，特别重要的是短时间点（50-150 ms）。我们表明，结构动力学和稳定性的信息可以测量伸展弱稳定的多肽在小肽和一个大的酶，糖原磷酸化酶的局部区域。
Amide hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for analyzing the conformational dynamics of proteins in a solution. Current conventional methods have a measurement limit starting from several seconds and are solely reliant on the speed of manual pipetting or a liquid handling robot. Weakly protected regions of polypeptides, such as in short peptides, exposed loops and intrinsically disordered the protein exchange on the millisecond timescale. Typical HDX methods often cannot resolve the structural dynamics and stability in these cases. Numerous academic laboratories have demonstrated the considerable utility of acquiring HDX-MS data in the sub-second regimes. Here, we describe the development of a fully automated HDX-MS apparatus to resolve amide exchange on the millisecond timescale. Like conventional systems, this instrument boasts automated sample injection with software selection of labeling times, online flow mixing and quenching, while being fully integrated with a liquid chromatography–MS system for existing standard “bottom-up” workflows. HDX-MS’s rapid exchange kinetics of several peptides demonstrate the repeatability, reproducibility, back-exchange, and mixing kinetics achieved with the system. Comparably, peptide coverage of 96.4% with 273 peptides was achieved, supporting the equivalence of the system to standard robotics. Additionally, time windows of 50 ms–300 s allowed full kinetic transitions to be observed for many amide groups; especially important are short time points (50–150 ms) for regions that are likely highly dynamic and solvent- exposed. We demonstrate that information on structural dynamics and stability can be measured for stretches of weakly stable polypeptides in small peptides and in local regions of a large enzyme, glycogen phosphorylase.
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影响因子：
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影响因子：
2.3
作者：
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通讯作者：
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