Probing non-covalent membrane protein-lipid interactions using sheath liquid capillary electrophoresis-native top-down mass spectrometry.

Probing non-covalent membrane protein-lipid interactions remains challenging due to the high hydrophobicity and aggregation propensity of membrane proteins. Here, we present a sheath liquid capillary electrophoresis-native top-down mass spectrometry (CE-nTDMS) platform that enables reproducible infusion of aquaporin Z-cardiolipin assemblies and preserves their non-covalent electrostatic interactions during top-down fragmentation. This workflow generated a diagnostic fragment ion that localizes a lipid-binding region within the protein backbone. Notably, charge-reduced precursor fragment ions of the lipid-bound assembly exhibited asymmetrical isotopic distributions following collisional activation, while corresponding apo precursors displayed symmetrical isotopic distributions. These findings demonstrate the utility of CE-nTDMS for reliable analysis of membrane protein-lipid assemblies and provide new insight into the behavior of lipid-bound fragments during collisional activation.