Complete Extraction of Protein Dynamics Information in Hydrogen/Deuterium Exchange Mass Spectrometry Data

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has been used to study protein conformation and conformational dynamics. A continuous labeling experiment, followed by proteolytic digestion and MS analysis, generates a large amount of data, containing information on protein conformation and conformational dynamics. Lacking appropriate computational methods, information hidden inside the isotope distribution is often omitted and not extracted. In this work, a computational model is described to simulate the determined isotope pattern for each proteolytic peptide at each labeling time. Optimizing the model with experimental data yields conformational protection as well as protein unfolding/folding kinetics. With this method, complete extraction of protein dynamics information in the HDX–MS data is achieved. Information derived from the method is reliable as the model is mostly based on first-principles with very few assumptions. It is demonstrated that the protein dynamics information extracted from one or two labeling time points approaches or exceeds the information derived from an entire deuterium uptake time course by the traditional method. Application of the computational method to an IgG1 antibody under mild denaturing conditions indicates that the unfolding of each immunoglobulin domain can be explained by a simple two-state unfolding process, with different unfolding rate for each domain.氢氘交换（Hydrogen/deuterium exchange, HDX）质谱法（mass spectrometry, MS）现已广泛应用于蛋白质构象及构象动力学的研究。通过连续标记实验，随后结合蛋白水解酶切与质谱分析，可获取大量蕴含蛋白质构象与构象动力学信息的数据集。然而由于缺乏适配的计算方法，同位素分布中隐藏的有效信息常被忽略而未被提取利用。本研究提出一种计算模型，用于模拟每个标记时间点下各蛋白水解肽段的同位素分布特征。通过实验数据对该模型进行优化，可同时获得蛋白质的构象保护信息与蛋白折叠/解折叠动力学参数。借助该方法，可实现HDX-MS数据中蛋白质动态信息的完全提取。由于该模型主要基于第一性原理且仅引入极少假设，由此得到的信息具备较高可靠性。研究表明，仅通过1或2个标记时间点提取的蛋白质动态信息，其可靠性已接近甚至超越传统方法通过完整氘摄取时间序列所获得的信息。将该计算方法应用于温和变性条件下的IgG1抗体后发现，每个免疫球蛋白结构域的解折叠过程均可通过简单的两态解折叠模型解释，且各结构域的解折叠速率存在差异。