Measuring Membrane Protein-Lipid Interactions in Nanodiscs with Native Mass Spectrometry

Abstract

Native mass spectrometry (MS), which uses nondenaturing ionization to preserve noncovalent complexes for mass analysis, has proven to be powerful for characterizing membrane protein complex stoichiometry and interactions. Membrane proteins are traditionally solubilized using detergent micelles prior to native MS. Collisional activation is employed in the mass spectrometer to remove bound detergent and yield the membrane protein, potentially in complex with bound lipids. However, this method of solubilization can be problematic as detergents may distort the native-like structure and function of membrane proteins. Furthermore, the collisional activation required for detergent removal can lead to the loss of noncovalent interactions within fragile complexes. Lipoprotein nanodiscs provide a more natural lipid bilayer environment for delivering membrane proteins for native MS, but previous measurements have been limited by the moderate stability of nanodiscs in the gas phase. It is difficult to preserve the intact membrane protein-nanodisc complex for native MS but also difficult to efficiently eject membrane proteins from nanodiscs. To tune the stability of nanodiscs, we used chemical additives known to modulate the charge acquired during electrospray ionization (ESI). Two membrane proteins, the trimeric ammonium transporter AmtB and tetrameric aquaporin ApqZ, were reconstituted into nanodiscs for native MS. Employing charge manipulation reagents and different ionization polarities allowed us to either capture the intact membrane protein-nanodisc complex or eject the membrane protein with few bound lipids. We are currently investigating the selectivity of lipid binding to membrane proteins ejected from nanodiscs of mixed lipid compositions. Preliminary results show that distinct lipid shells corresponding to different types of protein-lipid interactions are preferentially enriched in either zwitterionic or anionic lipids. Ultimately, we expect our novel methods combining native MS and nanodiscs will provide unique insights into membrane protein-lipid interactions in model lipid bilayers.