Construction of a High-Throughput Hydrogen Deuterium Exchange Mass Spectrometry Platform

高通量氢氘交换质谱平台的构建

• 批准号: BB/V019635/1
• 负责人: Glenn Masson
• 金额: $ 95.06万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV019635%2F1
• 关键词: Construction; Throughput; Hydrogen; Deuterium; Exchange

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a structural biology technique which excels in the investigation of protein interactions. All proteins have amide groups along their polypeptide backbone which routinely undergo a phenomenon of solvent exchange - where the hydrogen of the amide swaps with the hydrogen of the surrounding solvent, typically water. The rate of this exchange is inextricably linked to the structural environment of that amino acid on the polypeptide backbone. Areas of protein which are rigid, densely packed and highly structured have a much slower solvent exchange rate than disordered, flexible and loosely packed parts of the protein. Additionally, the creating of new bonds through the creation of new interaction interfaces, whether that be protein: protein interactions, or protein: drug, protein: membrane, or protein: nucleic acid, will all alter the solvent exchange rate.The measurement solvent exchange is achieved through the exposure of a protein to a deuterated solvent. When a solvent exchange event occurs, the protein's hydrogen atom is swapped for deuterium, and thus the mass of the amino acid is altered. Using mass spectrometry equipment, we can detect this increase in mass, and by incubating the protein with deuterium for multiple time points, we can measure the solvent exchange rate. The solvent exchange rate can then be manipulated by e.g. adding a drug which interacts with the protein, and determine where on the protein the solvent exchange rate is altered, thus locate where on a protein a drug, binding partner, or lipid membrane is interacting. Determining how proteins interact with other entities can provide insights into their mechanisms, and can rationalise how diseases may alter their structural properties and impact their function. Likewise, determining where drugs interact with proteins can guide their development and inform how they may be altered to be more potent, or have fewer side-effects. HDX-MS has numerous advantages over other technologies which may also provide structural insights into how proteins interact with other agents. For example, the technique of x-ray crystallography is highly dependent on having a rigid protein structure, so proteins which are intrinsically disordered cannot be investigated. Another technology, cryo-electron microscopy, can overcome some of these limitations, but it is a very slow technique for screening molecules.This proposal is for a LEAP HDX Extended System automated sample handling robot, attached to a Waters Synapt XS HDMS Mass Spectrometer. Together these two pieces of equipment work seamlessly to produce the highest quality of HDX-MS data, allowing for the most complex and cutting-edge applications of the technique, while also ensuring the greatest efficiency of mass spectrometry capacity. The LEAP HDX Extended System allows for highly reproducible data collection and sample preparation. HDX-MS experiments are extremely sensitive to temperature fluctuations and incubation timing, making sample preparation ideally conducted by robotics. Additionally, the work-flow of chromatography involves multiple steps, again ideally conducted through automation to ensure reproducibility. Furthermore, the nature of HDX-MS experiments requires numerous samples being analysed individually on a single instrument, and a robotically handled sample injection system allows for a maximisation of useful data collection, especially through the night and over weekends. The Waters Synapt XS HDMS Mass Spectrometer is well suited for HDMS. Due to the temperature constraints on HDX samples, the ability to resolve peptides by chromatography is limited. The ion-mobility ability of the Synapt XS compensates for this when analysing complex mixtures of proteins. Additionally, the ability to use alternative fragmentation methods allows for more cutting edge HDX-MS experiments to be conducted which increase the resolution of the technique.

氢-氢交换质谱仪(HDX-MS)是一种结构生物学技术，在研究蛋白质相互作用方面表现突出。所有蛋白质的多肽骨架上都有酰胺基团，这通常会经历一种溶剂交换现象--酰胺的氢与周围溶剂的氢交换，通常是水。这种交换的速度与多肽骨架上氨基酸的结构环境有着千丝万缕的联系。与蛋白质的无序、柔性和松散的包装部分相比，刚性、密集和高度结构化的蛋白质区域的溶剂交换速率要慢得多。此外，通过创建新的相互作用界面来创建新的键，无论是蛋白质：蛋白质相互作用，还是蛋白质：药物，蛋白质：膜，或蛋白质：核酸，都将改变溶剂交换速率。当发生溶剂交换事件时，蛋白质的氢原子被换成重氢，从而改变了氨基酸的质量。使用质谱仪，我们可以检测到这种质量的增加，通过将蛋白质与氘孵育多个时间点，我们可以测量溶剂交换率。然后，可以通过例如添加与蛋白质相互作用的药物来操纵溶剂交换率，并确定在蛋白质上哪里改变了溶剂交换率，从而定位药物、结合伙伴或脂膜在蛋白质上相互作用的位置。确定蛋白质如何与其他实体相互作用可以提供对其机制的洞察，并可以使疾病如何改变其结构属性和影响其功能合理化。同样，确定药物与蛋白质相互作用的位置可以指导它们的发展，并告知如何改变它们以使其更有效或副作用更少。与其他技术相比，HDX-MS具有许多优势，这也可能为蛋白质如何与其他试剂相互作用提供结构洞察力。例如，X射线结晶学技术高度依赖于具有刚性的蛋白质结构，因此无法研究本质上无序的蛋白质。另一种技术，冷冻电子显微镜，可以克服其中的一些限制，但它在筛选分子方面是一项非常缓慢的技术。这项提议是为了一个LEAP HDX扩展系统自动样品处理机器人，连接到Waters Synapt XS HDMS质谱仪。这两台设备无缝协作，以产生最高质量的HDX-MS数据，允许该技术最复杂和最尖端的应用，同时还确保最高效率的质谱学能力。LEAP HDX扩展系统允许高度可重复性的数据收集和样品制备。HDX-MS实验对温度波动和孵化时间极其敏感，因此由机器人进行样品制备是理想的。此外，层析的工作流程涉及多个步骤，同样理想的是通过自动化进行，以确保重现性。此外，HDX-MS实验的性质要求在一台仪器上单独分析大量样品，机器人操作的样品注入系统允许最大限度地收集有用的数据，特别是在夜间和周末。Waters Synapt XS HDMS质谱仪非常适合HDMS。由于HDX样品的温度限制，通过层析分离多肽的能力是有限的。在分析复杂的蛋白质混合物时，Synapt XS的离子迁移能力弥补了这一点。此外，使用替代碎裂方法的能力允许进行更多尖端的HDX-MS实验，从而提高技术的分辨率。