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

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.