Probing protein–lipid interactions by hydrogen/deuterium exchange-mass spectrometry: advances, challenges and future directions

Abstract

Membrane proteins constitute over 30% of the human proteome and represent more than 60% of drug targets, making them of critical interest in pharmaceutical discovery. Membrane proteins function within complex lipid environments that actively regulate their structure, dynamics, and activity. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful approach for probing membrane protein dynamics in solution and native-like assemblies, including nanodiscs, SMALPs, and liposomes, paving the way to study integral membrane proteins within the context of living cells. In this perspective, we discuss HDX-MS analysis of membrane protein–lipid interactions, particularly key challenges such as lipid-induced ion suppression and chromatographic interference, and highlight recent advances in lipid removal strategies, subzero chromatography, mass spectrometry/ion mobility, and the integration of molecular dynamics simulations and artificial intelligence. We further emphasise the synergy of HDX-MS with lipidomics and native MS to better understand the interplay between lipid composition, binding stoichiometry, and structural dynamics. Together, these approaches establish an emerging multidimensional framework for understanding membrane protein–lipid interplay under physiologically relevant conditions.