Department of Chemistry, University of Calgary, Calgary, Alberta, Canada.
J Am Soc Mass Spectrom. 2013 Jul;24(7):1006-15. doi: 10.1007/s13361-013-0639-4. Epub 2013 May 7.
Hydrogen/deuterium exchange (HDX) methods generate useful information on protein structure and dynamics, ideally at the individual residue level. Most MS-based HDX methods involve a rapid proteolytic digestion followed by LC/MS analysis, with exchange kinetics monitored at the peptide level. Localizing specific sites of HDX is usually restricted to a resolution the size of the host peptide because gas-phase processes can scramble deuterium throughout the peptide. Subtractive methods may improve resolution, where deuterium levels of overlapping and nested peptides are used in a subtractive manner to localize exchange to smaller segments. In this study, we explore the underlying assumption of the subtractive method, namely, that the measured back exchange kinetics of a given residue is independent of its host peptide. Using a series of deuterated peptides, we show that secondary structure can be partially retained under quenched conditions, and that interactions between peptides and reversed-phase LC columns may both accelerate and decelerate residue HDX, depending upon peptide sequence and length. Secondary structure is induced through column interactions in peptides with a solution-phase propensity for structure, which has the effect of slowing HDX rates relative to predicted random coil values. Conversely, column interactions can orient random-coil peptide conformers to accelerate HDX, the degree to which correlates with peptide charge in solution, and which can be reversed by using stronger ion pairing reagents. The dependency of these effects on sequence and length suggest that subtractive methods for improving structural resolution in HDX-MS will not offer a straightforward solution for increasing exchange site resolution.
氢/氘交换 (HDX) 方法可提供有关蛋白质结构和动力学的有用信息，理想情况下是在单个残基水平上。大多数基于 MS 的 HDX 方法涉及快速蛋白水解消化，然后进行 LC/MS 分析，并在肽水平监测交换动力学。局部特定的 HDX 位点通常仅限于宿主肽的分辨率大小，因为气相过程可以使整个肽的氘化混乱。减去方法可能会提高分辨率，其中重叠和嵌套肽的氘水平以减去方式用于将交换定位到更小的片段。在这项研究中，我们探讨了减去方法的基本假设，即给定残基的测量回交换动力学与其宿主肽无关。使用一系列氘化肽，我们表明在猝灭条件下可以部分保留二级结构，并且肽与反相 LC 柱之间的相互作用可以加速和减缓残基 HDX，这取决于肽序列和长度。二级结构是通过柱相互作用在具有结构溶液倾向的肽中诱导的，这会使 HDX 速率相对于预测的无规卷曲值减慢。相反，柱相互作用可以将无规卷曲肽构象定向以加速 HDX，其程度与溶液中肽的电荷相关，并且可以通过使用更强的离子配对试剂来逆转。这些效应对序列和长度的依赖性表明，用于提高 HDX-MS 结构分辨率的减去方法不会为提高交换位点分辨率提供简单的解决方案。
