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

氢/氘交换和离子淌度质谱支撑蛋白质相互作用研究

• 批准号: BB/L015048/1
• 负责人: Perdita Barran
• 金额: $ 63.58万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL015048%2F1
• 关键词: Hydrogen; Deuterium; Exchange; Ion; Mobility

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.

该提案将资助质谱仪和相关的入口系统，用于分析蛋白质结构，动力学和相互作用。该仪器资源将由曼彻斯特大学工程与物理科学学院、生命科学学院、医学与人文科学学院的研究小组与来自其他5所大学的学术伙伴以及两个研究理事会研究所合作使用。它也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伙伴。所要求的仪器是Waters公司最近开发的HDX-MS平台，这是唯一可用于HDX-MS研究的商用完整系统。LC/MS和自动化的创新与软件相结合，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学院第一个自动化HDXMS离子迁移能力仪器。我们将遵循完整蛋白质分析的程序，首先在蛋白质和蛋白质复合物的天然条件下进行离子迁移质谱（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，导致离子以与它们的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，有可能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。该读数将告知酶的构象动力学，以及蛋白质与小的“类药物”分子和其他蛋白质聚集时相互作用时形状的变化。差分氢/氘交换耦合质谱已成为表征蛋白质构象变化的一种敏感技术。这两种技术的结合是极其偶然的，因为一个氢原子与一个氘原子的有利交换导致质量增加一个。我们筛选蛋白质构象的第二部分将使用HDX-MS测量的质量增加来“快照”特定状态下的给定蛋白质，当与酶消化的数据相结合时，可以提供残留物水平上构象变化的细节，这种方法类似于核磁共振，但使用的材料要少得多。基于英格兰人的开创性工作，有可能预测多肽中氨基酸的内禀酰胺氢交换率。这个交换速率受氢键的影响；测量给定酰胺氢的这种性质是探测蛋白质结构和动力学的一种极好的方法。交换速率受pH值和温度的影响，必须精确控制以获得有用的信息。在典型的HDXMS工作流程中，将目标蛋白孵育一组预定的时间点，在一定范围内的pH值或与潜在的结合伙伴一起，在每个反应结束时通过快速降低pH值和温度来淬灭HDX反应，然后消化蛋白质，并通过UPLC接口引入质谱仪。对于每个消化的多肽，事先知道的序列允许氘摄取百分比作为时间的函数绘制。使用ETC或ECD可以精确定位交换的氨基酸。对于蛋白质的每一种状态的氘摄取信息可以映射到序列或结构，如果可用的话。这种方法也将应用于完整的蛋白质，并适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。

项目成果

Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding.

• DOI: 10.1038/ncomms12163
• 发表时间: 2016-07-15
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Beveridge R;Migas LG;Payne KAP;Scrutton NS;Leys D;Barran PE
• 通讯作者: Barran PE

Relating gas phase to solution conformations: Lessons from disordered proteins.

• DOI: 10.1002/pmic.201400605
• 发表时间: 2015-08
• 期刊: Proteomics
• 影响因子: 3.4
• 作者: Beveridge R;Phillips AS;Denbigh L;Saleem HM;MacPhee CE;Barran PE
• 通讯作者: Barran PE

Validating Differential Volatilome Profiles in Parkinson's Disease

验证帕金森病的差异挥发组谱

• DOI: 10.26434/chemrxiv.12525323
• 发表时间: 2020
• 作者: Barran P

Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclin A

离子淌度质谱法测量 p27 及其结构域的构象景观，以及如何在与 Cdk2/cyclin A 相互作用时对其进行调节

• DOI: 10.1002/ange.201812697
• 发表时间: 2019
• 期刊: Angewandte Chemie
• 作者: Beveridge R

Quantitative data describing the impact of the flavonol rutin on in-vivo blood-glucose and fluid-intake profiles, and survival of human-amylin transgenic mice.

• DOI: 10.1016/j.dib.2016.11.077
• 发表时间: 2017-02
• 期刊: DATA IN BRIEF
• 影响因子: 1.2
• 作者: Aitken, Jacqueline F;Loomes, Kerry M;Riba-Garcia, Isabel;Unwin, Richard D;Prijic, Gordana;Phillips, Ashley S;Phillips, Anthony R J;Wu, Donghai;Poppitt, Sally D;Ding, Ke;Barran, Perdita E;Dowsey, Andrew W;Cooper, Garth J S
• 通讯作者: Cooper, Garth J S