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

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.