Analysis of Mixed Lipid Nanodiscs and Viral Capsids with Native Mass Spectrometry and Charge Detection-Mass Spectrometry

Natural cell membranes are diverse and complex amphipathic barriers with thousands of different lipids that play multiple physiological functions. How membrane protein structure and function are affected by different lipids in the cell membrane is understudied. Furthermore, the lipid bilayer composition can change with age, disease, and diet;how these changes affect membrane protein structure and their link to disease and pathology are unknown. Measuring how different lipid interactions affect membrane protein and peptide structure is challenging because they require a hydrophobic lipid environment to be stable. To address this challenge, this dissertation details methods to assemble lipid bilayer mimetics called nanodiscs with multiple lipids that are resolvable by native mass spectrometry (MS). This allows us to retain noncovalent interactions and measure the oligomeric state of incorporated peptides or proteins in intact mixed lipid nanodiscs. Furthermore, these methods are applied to the characterization of gene therapy delivery systems termed adeno-associated viral (AAV) capsids.Previously, native MS of nanodiscs was limited to one or two lipid systems because of the added polydispersity in the mass spectra caused by the addition of a new lipid. To resolve nanodiscs with multiple incorporated lipids, we chose lipids that were integer or fraction multiples of each other, so that their m/z peaks overlap constructively. Using this mass resonance approach, we resolved two lipid nanodisc systems with sterols, monosialotetrahexosyl-ganglioside (GM1), and cardiolipin. Then, we assembled and resolved model mammalian, bacterial, and mitochondrial nanodiscs with up to 4 different phospholipids, which were useful for characterizing polydisperse lipid interactions with LL-37, which is a potential therapeutic for antibiotic resistant bacteria. Moving to more complex lipid systems, we investigated nanodiscs made from commercially available natural lipid extract that provide