EXPANDING THE APPLICATION OF NANODISCS FOR NATIVE MASS SPECTROMETRY TO STUDY BIOMOLECULES

Abstract

Membrane proteins mediate critical physiological roles which establishes them as attractive therapeutic targets. However, membrane protein interactions have proven difficult to study in a native lipid environment given their structural lability, transient nature, as well as the lack of lipid bilayer platforms to study protein-lipid interactions. Therefore, nanodiscs were developed for use in conjunction with native mass spectrometry to improve membrane protein analysis. Nanodiscs serve as a tool that provide a stable, biologically relevant lipid bilayer platform to solubilize membrane proteins. They are favorable given their more native state than detergent micelles and bicelles. Native mass spectrometry of membrane protein-nanodisc complexes allows for further investigation and characterization of membrane protein-lipid interactions. Previously, native mass spectrometry of nanodiscs was limited by the lack of nanodisc controllability. The occasional instability of nanodiscs makes it difficult to preserve entire nanodisc complexes with embedded membrane proteins and the inability to significantly destabilize nanodisc complexes limits ejection of biomolecules. We demonstrate that electrospray ionization conditions as well as charge manipulation chemical additives modulate nanodisc stability during native mass spectrometry. The significant difference in stability of nanodiscs with various chemical additives and electrospray ionization conditions expands the application of native mass spectrometry of nanodiscs and opens new avenues for study. Former studies have synthesized nanodiscs containing one or two prominent phospholipids to study membrane proteins and other peptide interactions. Unfortunately, these nanodiscs are lacking the complexity of a natural polydisperse lipid bilayer. To better model biological membranes and provide a more biologically consistent environment for the study of membrane proteins, we developed nanodiscs which mimic mammalian, bacterial and mitochondrial lipid compositions, with as many as four different phospholipids, whose structure was then determined with native mass spectrometry. We applied our approach to successfully characterize the incorporation of the human antimicrobial peptide LL-37 in single lipid versus bacterial nanodiscs. The development of model membrane nanodiscs provides a more in depth understanding of the assembly of complex nanodiscs and expands the available tools for studying lipoproteins in model biological membranes. Additionally, native mass spectrometry allows for the characterization of high-mass complexes and evaluation of molecular interactions but usually requires resolution of the different charge states produced by electrospray ionization, a process that is difficult to accomplish for extremely polydisperse samples. These samples tend to have overlapping charge states which leads to unresolvable spectra. Charge detection mass spectrometry (CD-MS) aims to address these challenges posed by conventional native mass spectrometry by simultaneously measuring the charge and m/z of isolated ions. There is occasional charge state uncertainty that limits the resolution of spectra, but this is addressed by the development of UniDecCD software for computational deconvolution of CD-MS data. UniDecCD improves the resolution of large heterogeneous samples including megadalton viral capsids and heterogeneous nanodiscs made from natural lipid extracts. Therefore, we provide a new computational tool for CD-MS data analysis as well as expand the application of nanodiscs to studying natural lipid extracts and extremely large, polydisperse biomolecular complexes. Overall, we discuss various ways to expand the application of nanodiscs for native mass spectrometry and the study of biomolecular structures.