Interfacing Nanodiscs with Native Mass Spectrometry to Study Membrane Protein-Lipid Interactions

Membrane proteins play vital roles in many biological processes and represent the majority of drug targets. The structural and functional integrity of membrane proteins can be heavily influenced by their surrounding lipid environment. However, membrane protein-lipid interactions are challenging to study in lipid bilayers due to their transient and polydisperse nature. Nanodiscs provide a versatile and robust membrane system for solubilizing membrane proteins in a native-like lipid bilayer. This dissertation describes the application of nanodiscs and native mass spectrometry to investigate the selectivity of membrane protein-lipid interactions. Charge manipulation additives and electrospray ionization conditions described in this dissertation allow membrane protein nanodiscs to be stabilized or destabilized during native mass spectrometry. Supercharging reagents in negative mode stabilized nanodiscs containing the ammonium transporter (AmtB) or aquaporin (ApqZ) and facilitated the measurement of membrane protein oligomeric state within an intact lipid bilayer. Conversely, supercharging reagents in positive mode ejected AmtB and AqpZ with tightly bound lipids, allowing the measurement of more specific interactions. To study how AmtB remodels its surrounding lipid environment, native mass spectrometry was performed for AmtB-lipid complexes following gas-phase ejection or solution-phase detergent extraction. Studies showed that AmtB is selective overall for POPG lipids but is enriched in POPC for tightly bound lipids. The mass spectrometry approaches detailed in this dissertation provide novel directions in using nanodiscs for characterizing membrane protein-lipid interactions in heterogeneous systems and are promising for applying nanodiscs in therapeutic development.