Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
Department of Biochemistry and Molecular Biology, Calgary, Alberta T2N 4N1, Canada.
Anal Chem. 2023 Mar 7;95(9):4421-4428. doi: 10.1021/acs.analchem.2c05003. Epub 2023 Feb 22.
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a rapidly growing technique for protein characterization in industry and academia, complementing the "static" picture provided by classical structural biology with information about the dynamic structural changes that accompany biological function. Conventional hydrogen deuterium exchange experiments, carried out on commercially available systems, typically collect 4-5 exchange timepoints on a timescale ranging from tens of seconds to hours using a workflow that can require 24 h or more of continuous data collection for triplicate measurements. A small number of groups have developed setups for millisecond timescale HDX, allowing for the characterization of dynamic shifts in weakly structured or disordered regions of proteins. This capability is particularly important given the central role that weakly ordered protein regions often play in protein function and pathogenesis. In this work, we introduce a new continuous flow injection setup for time-resolved HDX-MS (CFI-TRESI-HDX) that allows automated, continuous or discrete labeling time measurements from milliseconds to hours. The device is composed almost entirely of "off-the-shelf" LC components and can acquire an essentially unlimited number of timepoints with substantially reduced runtimes compared to conventional systems.
氢氘交换质谱（HDX-MS）是一种在工业和学术界中用于蛋白质特性分析的快速发展的技术，它通过提供有关伴随生物功能的动态结构变化的信息，补充了经典结构生物学提供的“静态”图像。在商业上可用的系统上进行的常规氢氘交换实验通常在数十秒到数小时的时间范围内收集 4-5 个交换时间点，使用可能需要 24 小时或更长时间的连续数据采集来进行三重复测量的工作流程。少数小组已经开发了用于毫秒时间尺度 HDX 的设置，从而能够对蛋白质中弱结构或无序区域的动态变化进行特征描述。鉴于弱有序蛋白质区域在蛋白质功能和发病机制中经常起核心作用，这种能力尤其重要。在这项工作中，我们引入了一种新的用于时间分辨 HDX-MS（CFI-TRESI-HDX）的连续流动注射装置，该装置允许从毫秒到小时自动、连续或离散地进行标记时间测量。该设备几乎完全由“现成”的 LC 组件组成，与传统系统相比，它可以在大大减少运行时间的情况下采集几乎无限数量的时间点。
