Pinpointing non-covalent lipid binding sites in membrane proteins via native top-down mass spectrometry

Pinpointing non-covalent lipid-binding sites remains a major challenge in structural biology. Lipids act as dynamic cofactors that regulate membrane protein folding, function, and drug interactions, yet their precise localization is often inaccessible. Here, we introduce a capillary electrophoresis–native top-down mass spectrometry (CE–nTDMS) platform that preserves and maps labile membrane protein–lipid interactions at residue-level resolution. Using Aquaporin Z, a tetrameric transmembrane protein, we demonstrate controlled subunit ejection, retention of intact phospholipid adduct, as well as localization of lipid-binding and post-translational modification (PTM) sites. Optimized desolvation voltage and collisional dissociation produced diagnostic holo fragment ions that pinpoint lipid–protein contact sites, defining complex stability and organization. These findings were validated using TDValidator software. This strategy establishes nTDMS as a chemical “microscope” for weak, non-covalent interactions, enabling structural elucidation of lipid-mediated modulation in membrane proteins at the residue level.