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

Abstract

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 the most native lipid environment to study membrane proteins. Because natural lipid extract nanodiscs have different lipid masses, they are unresolvable by conventional native MS. To resolve natural lipid extract nanodiscs, we used Orbitrap-based native charge detection mass spectrometry (CD-MS), which simultaneously measures the charge and m/z to get the mass. By using computational deconvolution, we improved the resolution of CD-MS for nanodiscs and AAV capsids. To improve the sensitivity of these native MS and CD-MS measurements, we chemically modified the surface of electrospray needles used for these experiments, which gave higher ion currents for AAVs and standard proteins. We then coupled CD-MS with variable temperature (VT)-MS to characterize the thermal unfolding of AAV capsids. We confirmed that filled capsids eject their cargo before completely unfolding, and VT-CD-MS provides a useful tool for measuring both the melting temperature and DNA ejection temperature of AAV capsids. Overall, the methods of this thesis detail the analysis of heterogenous complexes with native MS and CD-MS. The nanodisc platforms assembled and their characterization with native MS lay the groundwork to study how changes in the lipid bilayer composition affect pharmacologically relevant membrane peptides and proteins. Also, CD-MS deconvolution and VT-CD-MS offers a unique tool to quality control viral capsid-based therapies and will further gene therapy and vaccine therapeutic development.