Advancing Hydrogen/Deuterium Exchange Mass Spectrometry to Study Complex Systems

在化学部(CHE)和生物基础设施部(DBI)化学测量和成像计划的支持下，华盛顿大学的Miklos Guttman和他的团队正在努力推进氢/氢交换与质谱学(HDX-MS)相结合，以表征蛋白质结构。蛋白质是生物学的基石，因为它们几乎完成了生命所必需的所有生物过程。除了表征它们的结构外，了解它们在溶液中的行为也很重要。通常情况下，蛋白质在其功能过程中会采用不同的结构并在不同结构之间转换。氢/氢交换-质谱仪(HDX-MS)是少数几种成熟的能够监测蛋白质结构变化的方法之一。几十年来，HDX-MS的研究为蛋白质的功能提供了大量的洞察力，但HDX-MS的一个持续缺陷是它对变异非常敏感，两个不同的实验室在研究同一种蛋白质时很容易得到不同的结果。通过为这些实验制定内部标准，该项目寻求将HDX-MS测量标准化，以便所有研究人员可以在他们的数据之间进行可靠的比较。HDX-MS的一个关键优势是它可以监测蛋白质结构的亚群，但代替足够的数据分析软件，这些亚群经常被忽视。这项工作还将开发对HDX-MS数据进行稳健分析的软件工具，以便准确检测这些亚群，这些亚群通常与了解蛋白质功能高度相关。该团队还将为K-12学生提供外展机会，向他们介绍尖端分析技术的力量。古特曼研究小组使用HDX-MS研究了多种蛋白质。HDX-MS已成为研究蛋白质界面、直接测量蛋白质动力学和表征其他技术通常看不见的瞬时构象的广泛方法。HDX-MS的优势之一是能够分析广泛的蛋白质体系，包括糖蛋白、膜蛋白和大型蛋白质复合体，对于大多数结构技术来说，这些往往是具有挑战性的，如果不是不可能的话。值得注意的是，HDX-MS擅长于检测和表征蛋白质的多种构象共存时的情况。然而，仍然存在一些挑战，这些挑战往往限制了从HDX-MS研究中可靠获得的信息的范围，包括重复性差、对表观多构象群体的误解以及样品清理的技术限制。该项目旨在应对这些挑战，并通过以下方式扩大适用于HDX-MS的系统的信息水平和复杂性限制：1)开发基于咪唑的小分子作为可靠的内部标准，以准确测量溶液交换条件；2)开发一种内部标准，以准确报告反应中的氚含量百分比；3)建立一种计算工具，用于准确检测和分析包含多种构象的样品中的双峰质膜。总体而言，这些进展预计将使HDX-MS研究的重复性和信息量显著增加。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry (CHE) and the Division of Biological Infrastructure (DBI), Miklos Guttman and his group at the University of Washington are working to advance hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) for characterizing protein structure. Proteins are the cornerstone of biology as they carry out nearly all biological processes essential for life. Beyond characterizing their structures, it is important to understand their behavior in solution. Often proteins will adopt and transition between different structures through the course of their function. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is one of the few established methods that is capable of monitoring changes in protein structure. For decades HDX-MS studies have provided massive insight into how proteins function, but an ongoing shortcoming of HDX-MS is that it is highly susceptible to variations and two different laboratories can easily get different results when studying the same protein. By developing internal standards for these experiments this project seeks to standardize HDX-MS measurements so all researchers can make reliable comparisons among their data. One key strength of HDX-MS is that it can monitor subpopulations of protein structures, but in lieu of adequate software for data analysis, these subpopulations often go unnoticed. This work will also develop software tools for robust analysis of HDX-MS data so these subpopulations, which are often highly relevant to understanding protein function, are accurately detected. The team will also provide outreach opportunities for K-12 students to be introduced to the power of cutting-edge analytical technologies. The Guttman group uses HDX-MS to study many classes of proteins. HDX-MS has become a widespread approach for studying protein interfaces, directly measuring protein dynamics, and characterizing transient conformations that are often invisible to other techniques. Among the strengths of HDX-MS is the ability to analyze a broad range of protein systems including glycoproteins, membrane proteins, and large protein complexes that are often challenging, if not impossible, for most structural techniques. Notably, HDX-MS is proficient at detecting and characterizing when multiple conformations of a protein coexist. However, there remain some challenges that often limit the scope of information that is reliably obtained from HDX-MS studies including poor reproducibility, misinterpretation of apparent multiple conformational populations, and technical limitations with sample clean up. This project aims to address these challenges and expand the level of information and limits on complexity of systems amenable to HDX-MS through: 1) development of imidazolium-based small molecules to serve as robust internal standards to accurately measure solution exchange conditions; 2) development of an internal standard to accurately report percentage of deuterium content in a reaction; 3) establishment of a computation tool for accurately detecting and analyzing bimodal mass envelopes in samples containing multiple conformations. Overall, these developments are expected to make HDX-MS studies significantly more reproducible and informative.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.