Work

Practical Tips for the Application of HDX-MS to Membrane Proteins, Biomolecular Condensates, and Weak Protein Binders

Year: 2025

Type: article

Abstract: Hydrogen-deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful biophysical tool to study protein thermodynamics, dynamics, and function. Despite its increasing use, effective implementation of HDX-MS requires knowledge of protein chemistry, folding, liquid chromatography, MS, and automation. Whereas method development for soluble globular proteins is often easily generalizable, HDX-MS on more complex systems is challenging. Published method sections are usually concise, and available step-by-step protocols typically only provide generalized procedures. Here, we offer tips for applying HDX-MS to three complex systems: integral membrane proteins, biomolecular condensates, and weak protein binders. These practices have yielded high sequence coverage and high-quality data, allowing for free energy quantification and, importantly, mechanistic understanding of protein functions. We believe that sharing our methodologies will help others apply and develop HDX-MS methods for these and similar systems.

Source: Journal of the American Society for Mass Spectrometry

DOI: 10.1021/jasms.5c00067

Authors: Xiaoxuan Lin, Andrew V. Molina, Julia Shangguan, Ruofan Chen, Tobin R. Sosnick

Institution: University of Chicago

Language: English

Topic: Mass Spectrometry Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funders: National Heart, Lung, and Blood Institute, National Institute of General Medical Sciences, National Science Foundation Graduate Research Fellowship Program

Awards: G2589448798, G2861173083, G7542389844, G7697095618, Molecular and Cellular Biology Training Program