Hydrogen/Deuterium Exchange and Ion Mobility Mass Spectrometry to Underpin Research on Protein Interactions

这项提议将为质谱仪和相关的进样系统提供资金，用于分析蛋白质的结构、动力学和相互作用。该仪器资源将由曼彻斯特大学工程和物理科学、生命科学、医学和人文科学学院的研究小组与其他5所大学的学术合作伙伴以及两个研究委员会研究所合作使用。它还将提供给我们在工业生物技术、生物医药和农业食品部门的工业合作伙伴。所要求的仪器是Waters公司最近开发的HDX-MS平台，这是用于HDX-MS研究的唯一商业可用的完整系统。在LC/MS和自动化方面的创新，再加上软件，使HDMX成为表征蛋白质结构和相互作用的强大工具。这将是英国高等教育研究所的第一台自动化HDXMS离子移动性仪器。我们将遵循的完整蛋白质分析程序将首先对蛋白质和蛋白质复合体进行自然条件下的离子迁移质谱仪(IM-MS)。由于固有的蛋白质动力学、配体结合或蛋白质相互作用引起的构象变化将是可测量的。IM-MS记录了在充满惰性缓冲气体的弱电场的影响下，质量选择的离子穿过电池所需的时间。与缓冲气体的碰撞阻碍了离子的前进，再加上离子的电荷，导致离子以与其大小成正比的速度漂移。通过测量离子穿过细胞所需的时间，可以获得旋转平均横截面，这是一个粗略的结构参数，可以告诉我们任何给定分子的大小。这个读数将告知酶的构象动力学，以及蛋白质与小的“类药物”分子相互作用时的形状变化，以及当它们聚集时与其他蛋白质的相互作用。差示氢/氢交换与质谱学联用已成为表征蛋白质构象变化的一种灵敏技术。这两种技术的结合是非常偶然的，因为氢原子与重氢原子的有利交换导致质量增加1。蛋白质构象筛选的第二部分将使用HDX-MS测量的质量增加来‘快照’特定状态下的特定蛋白质，当与来自酶消化的数据相结合时，这将提供残基水平构象变化的细节，这一方法类似于核磁共振，但使用的材料要少得多。基于英格兰德的开创性工作，有可能预测多肽中氨基酸的固有酰胺氢交换率。这种交换率受氢键的影响；对于给定的酰胺氢，测量这种性质是探索蛋白质结构和动力学的一种极好的方法。汇率受pH值和温度的影响，必须精确控制这些因素才能获得有用的信息。在典型的HDXMS工作流程中，目标蛋白质孵育一组预定的时间点，并在一定范围的PHS上或与潜在的结合伙伴孵育，通过迅速降低pH和温度在每个反应结束时猝灭HDX反应，然后消化蛋白质，并通过UPLC接口将其引入质谱仪。对于每个被消化的多肽，事先知道的序列允许绘制出作为时间函数的吸氢率。使用ETC或ECD可以精确定位已交换的氨基酸。蛋白质每种状态的吸氢信息可以映射到序列或结构(如果有)。这种方法也将应用于完整的蛋白质，并可用于复杂的混合物分析，例如在食品中确定过敏反应的分子基础。

This proposal will fund a mass spectrometer and associated inlet system for the analysis of protein structure, dynamics and interactions. The instrument resource will be used by research groups from the Faculties of Engineering and Physical Sciences, Life Sciences, and Medicine and Human Sciences at the University of Manchester, in collaboration with academic partners from 5 other Universities, along with two research council Institutes. It also will be available to our industrial partners in industrial biotechnology, biomedicine and agri-food sectors. The requested instrument is an HDX-MS platform recently developed by Waters Corporation, the only commercially-available complete system for HDX-MS studies. Innovations in LC/MS and automation coupled with software position HDMX as a robust tool to characterise protein structure and interactions. This will be the first automated HDXMS ion mobility capable instrument in a UK Higher Education Institute. The procedure we will follow for intact protein analysis will first perform ion mobility mass spectrometry (IM-MS) from native conditions on proteins and protein complexes. Conformational change due to inherent protein dynamics, ligand binding, or protein interaction will be measurable. IM-MS records the time it takes mass selected ions to traverse a cell under the influence of a weak electric field filled with an inert buffer gas. Collisions with the buffer gas impede the progress of the ion, and this coupled with the charge of the ion, causes the ion to drift with a velocity that is proportional to their size. By measuring the time it takes ions to cross the cell, it is possible to obtain the rotationally averaged cross section, a coarse structural parameter that will tell us the size of any given molecule. This readout will inform on conformational dynamics for enzymes, and on the change in shape of proteins as they interact with small 'druglike' molecules and with other proteins as they aggregate. Differential hydrogen/deuterium exchange coupled with mass spectrometry has emerged as a sensitive technique to characterise changes in protein conformation. The marrying of these two techniques is extremely fortuitous, since the favorable exchange of a hydrogen atom for a deuterium atom results in a mass increase of one. The second part of our screen for protein conformation will use the mass increase measured by HDX-MS to 'snapshot' a given protein in a particular state, which when coupled with data from enzymatic digestion provides details of conformational changes at the residue level, an approach which is analogous to NMR, but uses far less material. Based on the pioneering work of Englander, it is possible to predict intrinsic amide hydrogen exchange rates for amino acid in a polypeptide. This exchange rate is influenced by hydrogen bonding; measuring this property for a given amide hydrogen is an excellent way to probe protein structure and dynamics. Exchange rates are influenced by pH and temperature, and these must be precisely controlled to gain useful information. In a typical HDXMS workflow, a target protein is incubated for a set of predetermined time points, and over a range of pHs or with a potential binding partner, the HDX reaction is quenched at the end of each of these reactions by rapidly lowering the pH and temperature, and the protein is then digested, and introduced via an UPLC interface to the mass spectrometer. For each digested polypeptide, prior knowledge of sequence allows the percentage deuterium uptake to be plotted as a function of time. The use of ETC or ECD allows the precise amino acid that has exchanged to be located. Deuterium uptake information for each state of the protein can be mapped to the sequence or to the structure if available. This approach will also be applied to intact proteins, and is amenable to complex mixture analysis for example in food stuffs to determine the molecular basis for an allergic response.