Peptide-Column Interactions and Their Influence on Back Exchange Rates in Hydrogen/Deuterium Exchange-MS
Peptide-Column Interactions and Their Influence on Back Exchange Rates in Hydrogen/Deuterium Exchange-MS
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DOI：
10.1007/s13361-013-0639-4
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发表时间：
2013-07-01
影响因子：
3.2
通讯作者：
Schriemer, David C.
中科院分区：
文献类型：
--
作者：
Sheff, Joey G.;Rey, Martial;Schriemer, David C.
氢/氘交换（HDX）方法产生关于蛋白质结构和动力学的有用信息，理想地在单个残基水平上。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，并在肽水平上监测交换动力学。定位HDX的特定位点通常限于宿主肽的大小的分辨率，因为气相过程可以在整个肽中扰乱氘。消减方法可以提高分辨率，其中重叠和嵌套肽的氘水平以消减方式用于将交换定位到较小的区段。在这项研究中，我们探讨的基本假设的减法，即，所测得的反交换动力学的一个给定的残基是独立的其主机肽。使用一系列的氘代肽，我们表明，二级结构可以部分保留在淬灭条件下，和肽和反相LC柱之间的相互作用可能会加速和减速残基HDX，这取决于肽序列和长度。二级结构是通过在具有溶液相结构倾向的肽中的柱相互作用诱导的，其具有相对于预测的无规卷曲值减慢HDX速率的效果。相反，柱相互作用可以定向随机卷曲肽构象以加速HDX，其程度与溶液中的肽电荷相关，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中结构分辨率的减法方法不会为提高交换位点分辨率提供直接的解决方案。
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.