Native Mass\nSpectrometry of Membrane Protein–Lipid\nInteractions in Different Detergent Environments

Native mass spectrometry\n(MS) reveals the role of specific lipids\nin modulating membrane protein structure and function. Membrane proteins\nsolubilized in detergents are often introduced into the mass spectrometer.\nHowever, detergents commonly used for structural studies, such as\ndodecylmaltoside, tend to generate highly charged ions, leading to\nprotein unfolding, thereby diminishing their utility in characterizing\nprotein–lipid interactions. Thus, there is a critical need\nto develop approaches to investigate protein–lipid interactions\nin different detergents. Here, we demonstrate how charge-reducing\nmolecules, such as spermine and trimethylamine-<i>N</i>-oxide,\nenable the opportunity to characterize lipid binding to the bacterial\nwater channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory\nprotein GlnK in different detergent environments. We find that protein–lipid\ninteractions not only are protein-dependent but also can be influenced\nby the detergent and type of charge-reducing molecule. AqpZ-lipid\ninteractions are enhanced in LDAO (<i>n</i>-dodecyl-<i>N</i>,<i>N</i>-dimethylamine-<i>N</i>-oxide),\nwhereas the interaction of AmtB-GlnK with lipids is comparable among\ndifferent detergents. A fluorescent lipid binding assay also shows\ndetergent dependence for AqpZ-lipid interactions, consistent with\nresults from native MS. Taken together, native MS will play a pivotal\nrole in establishing optimal experimental parameters that will be\ninvaluable for various applications, such as drug discovery as well\nas biochemical and structural investigations.