Retention Time Interpolation Enhances Peptide Mapping for HDX-MS.

IF 2.7 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Damon Griffiths, Juan P Rincon Pabon, Charlotte Guffick, Argyris Politis
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引用次数: 0

Abstract

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for studying protein structural dynamics. A critical step in the HDX-MS workflow is generating a peptide map from nondeuterated samples, which serves as the reference for identifying and monitoring peptides in subsequent deuterium-labeled experiments. Maximizing peptide identifications improves sequence coverage and redundancy, enhancing the information content and spatial resolution of the HDX-MS data. However, peptide identification is often limited by suboptimal peptide separation/fragmentation. In other proteomic workflows, longer liquid chromatography (LC) gradients are commonly used to improve the peptide identification by increasing resolution. However, in HDX-MS workflows, such gradients are generally incompatible due to time constraints imposed by deuterium/hydrogen back-exchange. To address this, we introduce a flexible workflow that uses long-gradients during initial peptide mapping, followed by retention time (RT) interpolation for application in subsequent short-gradient HDX-MS. By performing both long- and short-gradient peptide mapping, we used shared peptides to generate a regression model that predicts short-gradient RTs for all peptides identified in the long-gradient experiment. This enables the use of the richer peptide maps provided by long-gradient chromatography without compromising the deuterium retention. The method is implemented by RTinterpolator, a freely available R script compatible with widely used HDX analysis platforms that rely on reference RT values for peptide monitoring in deuterium-labeled data. By providing predicted RTs aligned to short gradients, RTinterpolator offers a practical, accessible, and instrument-independent way of increasing sequence coverage and redundancy in HDX-MS experiments, particularly for large or complex proteins susceptible to the limitations of short-gradient chromatography.

保留时间插值增强HDX-MS的肽图谱。
氢/氘交换质谱(HDX-MS)是研究蛋白质结构动力学的一种强有力的技术。HDX-MS工作流程中的一个关键步骤是从非氘化样品中生成肽图,该图可作为随后氘标记实验中识别和监测肽的参考。最大限度的多肽鉴定提高了序列覆盖率和冗余度,增强了HDX-MS数据的信息含量和空间分辨率。然而,肽的鉴定常常受到次优肽分离/片段化的限制。在其他蛋白质组学工作流程中,更长的液相色谱(LC)梯度通常用于通过增加分辨率来提高肽鉴定。然而,在HDX-MS工作流程中,由于氘/氢反交换的时间限制,这种梯度通常是不兼容的。为了解决这个问题,我们引入了一个灵活的工作流程,在初始肽映射期间使用长梯度,然后在随后的短梯度HDX-MS中应用保留时间(RT)插值。通过进行长梯度和短梯度肽映射,我们使用共享肽来生成一个回归模型,该模型可以预测长梯度实验中识别的所有肽的短梯度RTs。这使得使用长梯度色谱提供的更丰富的肽图而不影响氘保留。该方法由RTinterpolator实现,RTinterpolator是一个免费的R脚本,与广泛使用的HDX分析平台兼容,该平台依赖于参考RT值来监测氘标记数据中的肽。通过提供与短梯度相一致的预测RTs, RTinterpolator提供了一种实用的,可访问的,仪器独立的方法来增加HDX-MS实验中的序列覆盖和冗余,特别是对于易受短梯度色谱限制的大型或复杂蛋白质。
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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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