Strategies for Top–Down Hydrogen Deuterium Exchange-Mass Spectrometry: A Mini Review and Perspective

IF 1.9 3区 化学 Q3 BIOCHEMICAL RESEARCH METHODS
Joel B. Langford, Elizabeth Ahmed, Mulin Fang, Kellye Cupp-Sutton, Kenneth Smith, Si Wu
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引用次数: 0

Abstract

Hydrogen deuterium-exchange mass spectrometry (HDX-MS) is commonly used in the study of protein dynamics and protein interactions. By measuring the isotopic exchange of backbone amide hydrogens in solution, HDX-MS offers valuable structural insights into challenging biological systems. Traditional HDX-MS approaches utilize bottom–up (BU) proteomics, in which deuterated proteins are digested before MS analysis. BU-HDX enables the characterization of proteins with various sizes in simple protein mixtures or complex biological samples such as cell lysates. However, BU methods are inherently limited by the inability to resolve protein sub-populations arising from different protein conformations, such as those arising from post-translational modifications (PTMs). Alternatively, top–down (TD) HDX-MS detects the global deuterium uptake at the intact proteoform level, allowing direct probing of structural changes due to protein–protein interactions, PTMs, or conformational changes. Combining TD-HDX-MS with electron-based fragmentation techniques, such as electron capture dissociation (ECD) and electron transfer dissociation (ETD), has demonstrated the feasibility of studying intact protein interactions with amino acid-level resolution. Here, we present a brief overview of methodologies, limitations, and applications of TD-HDX-MS using direct infusion techniques and LC-based approaches. Furthermore, we conclude with a perspective on the future directions for TD-HDX-MS.

自上而下的氢氘交换质谱分析策略:小型回顾与展望
氢氘交换质谱(HDX-MS)常用于蛋白质动力学和蛋白质相互作用的研究。通过测量溶液中骨架酰胺氢的同位素交换,HDX-MS 为研究具有挑战性的生物系统提供了宝贵的结构见解。传统的 HDX-MS 方法采用自下而上(BU)蛋白质组学,即在质谱分析之前消化氚代蛋白质。BU-HDX 能够表征简单蛋白质混合物或复杂生物样本(如细胞裂解液)中不同大小的蛋白质。然而,BU 方法由于无法解析不同蛋白质构象(如翻译后修饰(PTM)产生的蛋白质)所产生的蛋白质亚群而受到固有的限制。另一种方法是自上而下(TD)HDX-MS,它能在完整蛋白质形式水平上检测全局氘吸收,从而直接探测蛋白质-蛋白质相互作用、PTM 或构象变化引起的结构变化。将 TD-HDX-MS 与电子捕获解离(ECD)和电子转移解离(ETD)等基于电子的碎裂技术相结合,证明了以氨基酸水平的分辨率研究完整蛋白质相互作用的可行性。在此,我们简要概述了使用直接注入技术和基于液相色谱的 TD-HDX-MS 方法、局限性和应用。最后,我们展望了 TD-HDX-MS 的未来发展方向。
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来源期刊
Journal of Mass Spectrometry
Journal of Mass Spectrometry 化学-光谱学
CiteScore
5.10
自引率
0.00%
发文量
84
审稿时长
1.5 months
期刊介绍: The Journal of Mass Spectrometry publishes papers on a broad range of topics of interest to scientists working in both fundamental and applied areas involving the study of gaseous ions. The aim of JMS is to serve the scientific community with information provided and arranged to help senior investigators to better stay abreast of new discoveries and studies in their own field, to make them aware of events and developments in associated fields, and to provide students and newcomers the basic tools with which to learn fundamental and applied aspects of mass spectrometry.
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