Characterizing the Lipid Annulus Surrounding Membrane Proteins with Native Mass Spectrometry of Nanodiscs

It is challenging to characterize the transient and heterogeneous interactions between membrane proteins and their surrounding lipids. High resolution structural biology techniques can detect tightly bound structural lipids but generally cannot detect weakly bound annular lipids. Spectroscopy techniques can detect relative proximity of lipids but require labels that may disrupt their behavior. By using nondenaturing ionization to preserve noncovalent complexes for mass analysis, native mass spectrometry (MS) provides a label-free strategy for detecting and characterizing protein-lipid complexes. However, conventional native MS of membrane proteins uses detergent micelles to solubilize the membrane protein, which limits analysis to complexes with only a handful of lipids bound. To allow analysis of a wide range of lipid interactions, we employ lipoprotein nanodiscs to deliver membrane proteins for native MS. Using optimized ionization conditions, we can measure the mass of the intact nanodisc, which allows direct measurement of the oligomeric state of the membrane protein within the lipid bilayer of the nanodisc. Collisional activation of nanodiscs in the gas phase releases membrane proteins surrounded by a complete annulus of lipids, which can be further activated to dissociate the weakly bound lipids in the annulus. We are developing new approaches using native MS of nanodiscs with binary combinations of lipids to characterize the composition and stoichiometry of this lipid annulus. Our ultimate goal is to develop a general method for characterizing protein-lipid interactions of varying strengths and specificities.