Advancing Hydrogen/Deuterium Exchange Mass Spectrometry to Study Complex Systems

University Of Washington, Seattle WA

Investigators

Abstract

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.