Macrocyclic Charge-Reducing Reagents for Native Mass Spectrometry of Membrane Proteins

Abstract Native mass spectrometry (MS) has become a powerful approach for characterizing membrane protein complexes by directly measuring noncovalent interactions with lipids, ligands, and other binding partners. However, membrane proteins often require collisional activation to remove detergent micelles prior to analysis, a process that can promote gas-phase unfolding, subunit dissociation, and loss of weakly bound ligands. Although chemical charge reduction can mitigate these effects, the repertoire of effective reagents for membrane protein native MS remains limited. Here, we demonstrate that macrocyclic compounds, including crown ethers and cyclen, effectively reduce charge in native MS across diverse membrane proteins and detergent environments. Among the compounds examined, cyclen exhibited the greatest charge-reducing potency, providing lower charge states while improving the retention of native oligomeric assemblies under collisional activation. Furthermore, macrocyclic charge reduction enhanced preservation of protein-lipid interactions, enabling more extensive characterization of lipid-bound states by native MS. Together, these findings expand the chemical toolbox for charge reduction and provide broadly applicable reagents for improving the native MS analysis of membrane proteins and their associated lipid interactions.