Advancing Hydrogen/Deuterium Exchange Mass Spectrometry to Study Complex Systems

推进氢/氘交换质谱研究复杂系统

• 批准号: 2304707
• 负责人: Miklos Guttman
• 金额: $ 45万
• 依托单位: University of Washington
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2304707&HistoricalAwards=false
• 关键词: Advancing; Hydrogen; Deuterium; Exchange; Mass

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.

在化学部（CHE）和生物基础设施部（DBI）的化学测量和成像项目的支持下，华盛顿大学的Miklos Guttman和他的团队正在努力推进氢/氘交换耦合质谱（HDX-MS）用于表征蛋白质结构。蛋白质是生物学的基石，因为它们执行几乎所有生命必需的生物过程。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，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/氘交换耦合质谱（HDX-MS）是为数不多的能够监测蛋白质结构变化的方法之一。几十年来，HDX-MS研究为蛋白质的功能提供了大量的见解，但HDX-MS的一个持续的缺点是它极易受到变异的影响，两个不同的实验室在研究相同的蛋白质时很容易得到不同的结果。通过制定这些实验的内部标准，该项目旨在标准化HDX-MS测量，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，但代替足够的软件进行数据分析，这些亚群往往被忽视。这项工作还将开发用于HDX-MS数据稳健分析的软件工具，以便准确检测这些通常与理解蛋白质功能高度相关的亚群。该团队还将为K-12学生提供拓展机会，让他们了解尖端分析技术的力量。Guttman小组使用HDX-MS来研究许多种类的蛋白质。HDX-MS已经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不可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系统，包括糖蛋白、膜蛋白和大型蛋白质复合物，这对于大多数结构技术来说通常是具有挑战性的，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的情况。然而，仍然存在一些挑战，这些挑战通常限制了从HDX-MS研究中可靠获得的信息范围，包括再现性差、对明显的多个构象群体的误解以及样品清理的技术限制。该项目旨在解决这些挑战，并通过以下方式扩大HDX-MS适用系统的信息水平和复杂性限制：1)开发咪唑基小分子，作为可靠的内部标准，以准确测量溶液交换条件；2)制定内部标准，以准确报告反应中氘含量的百分比；3)建立精确检测和分析多构象样品双峰质量包络的计算工具。总的来说，这些发展有望使HDX-MS研究的可重复性和信息量显著提高。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。