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

氢氘交换质谱（HDX-MS）是一种结构生物学技术，擅长研究蛋白质相互作用。所有的蛋白质都有沿着它们的多肽骨架的酰胺基团，其通常经历溶剂交换现象-其中酰胺的氢与周围溶剂（通常是水）的氢交换。这种交换的速率与多肽骨架上氨基酸的结构环境密不可分。刚性、密集堆积和高度结构化的蛋白质区域的溶剂交换速率比蛋白质的无序、柔性和松散堆积部分慢得多。此外，通过产生新的相互作用界面而产生新的键，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。当溶剂交换事件发生时，蛋白质的氢原子被交换为氘，因此氨基酸的质量被改变。使用质谱设备，我们可以检测到这种质量的增加，并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速率，并确定蛋白质上溶剂交换速率改变的位置，从而定位蛋白质上药物、结合配偶体或脂质膜相互作用的位置。确定蛋白质如何与其他实体相互作用可以深入了解其机制，并可以合理化疾病如何改变其结构特性并影响其功能。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，并告知它们如何改变以变得更有效或副作用更少。HDX-MS与其他技术相比具有许多优势，这些技术还可以提供蛋白质如何与其他试剂相互作用的结构见解。例如，X射线晶体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，可以克服这些限制，但它是一个非常缓慢的技术筛选molecules.This建议是一个LEAP HDX扩展系统自动样品处理机器人，连接到沃茨Synapt XS HDMS质谱仪。这两台设备可以无缝地协同工作，产生最高质量的HDX-MS数据，从而实现该技术最复杂和最前沿的应用，同时确保质谱分析能力的最大效率。LEAP HDX扩展系统允许高度可重复的数据收集和样品制备。HDX-MS实验对温度波动和孵育时间非常敏感，因此理想情况下可以通过机器人进行样品制备。此外，色谱的工作流程涉及多个步骤，理想情况下通过自动化进行，以确保重现性。此外，HDX-MS实验的性质需要在单个仪器上单独分析大量样品，而机器人操作的样品注射系统可以最大限度地收集有用的数据，特别是在夜间和周末。沃茨Synapt XS HDMS质谱仪非常适合HDMS。由于HDX样品的温度限制，通过色谱法解析肽的能力有限。Synapt XS的离子迁移能力在分析复杂的蛋白质混合物时弥补了这一点。此外，使用替代裂解方法的能力允许进行更多尖端HDX-MS实验，这增加了该技术的分辨率。

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.