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

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

Lipids and Small Molecules Affect α-synuclein Association and Disruption of Nanodiscs

Lipid membranes have recently been implicated in protein misfolding and disease etiology, including for α-synuclein and Parkinson’s Disease. However, it is challenging to study the intersection of protein complex formation, membrane interactions, and bilayer disruption simultaneously. In particular, the efficacies of small molecule inhibitors for toxic protein aggregation are not well understood. Here, we used native mass spectrometry in combination with lipid nanodiscs to study α-synuclein-membrane interactions. α-synuclein did not interact with zwitterionic DMPC lipids but interacted strongly with anionic DMPG lipids, eventually leading to membrane disruption. Unsaturated POPG lipid nanodiscs were also prone to bilayer disruption, releasing α-synuclein:POPG complexes. Interestingly, the fibril inhibitor, (-)-epigallocatechin gallate (EGCG), prevented membrane disruption but did not prevent the incorporation of α-synuclein into nanodisc complexes. Thus, although EGCG inhibits fibrilization, it does not inhibit α-synuclein from associating with the membrane

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry

Native mass spectrometry (MS) with nanodiscs is a promising technique for characterizing membrane protein and peptide interactions in lipid bilayers. However, prior studies have used nanodiscs made of only one or two lipids, which lack the complexity of a natural lipid bilayer. To better model specific biological membranes, we developed model mammalian, bacterial, and mitochondrial nanodiscs with up to four different phospholipids. Careful selection of lipids with similar masses that balance the fluidity and curvature enabled these complex nanodiscs to be assembled and resolved with native MS. We then applied this approach to characterize the specificity and incorporation of LL-37, a human antimicrobial peptide, in single lipid nanodiscs versus model bacterial nanodiscs. Overall, development of these model membrane nanodiscs reveals new insights into the assembly of complex nanodiscs and provides a useful toolkit for studying membrane protein, peptide, and lipid interactions in model biological membranes

Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry

Native mass spectrometry (MS) and charge detection-mass spectrometry (CD-MS) have become versatile tools for characterizing a wide range of proteins and macromolecular complexes. Both commonly use nano-electrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb to the glass, which may lower sensitivity and limit the quality of the data. To improve the sensitivity of native MS and CD-MS, we modified the surface of nESI needles with inert surface modifiers, including polyethylene-glycol. We found that the surface modification improved the signal intensity for native MS of proteins and for CD-MS of adeno-associated viral capsids. These surface modified needles provide a simple and inexpensive method for improving the sensitivity of challenging analytes

UniDecCD: Deconvolution of Charge Detection-Mass Spectrometry Data

Native mass spectrometry (MS) has become a versatile tool for characterizing high-mass complexes and measuring biomolecular interactions. Native MS usually requires resolution of different charge states produced by electrospray ionization to measure the mass, which is difficult for highly heterogeneous samples that have overlapping and unresolvable charge states. Charge detection-mass spectrometry (CD-MS) seeks to address this challenge by simultaneously measuring the charge and m/z for isolated ions. However, CD-MS often shows uncertainty in the charge measurement that limits the resolution. To overcome this charge state uncertainty, we developed UniDecCD (UCD) software for computational de-convolution of CD-MS data, which significantly improves the resolution of CD-MS data. Here, we describe the UCD algorithm and demonstrate its ability to improve CD-MS resolution of proteins, megadalton viral capsids, and heterogeneous nanodiscs made from natural lipid extracts. UCD provides a user-friendly interface that will increase the accessibility of CD-MS technology and provide a valuable new computational tool for CD-MS data analysis

Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry

Native mass spectrometry (MS) and charge detection-mass spectrometry (CD-MS) have become versatile tools for characterizing a wide range of proteins and macromolecular complexes. Both commonly use nanoelectrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb to the glass, which may lower sensitivity and limit the quality of the data. To improve the sensitivity of native MS and CD-MS, we modified the surface of nESI needles with inert surface modifiers, including polyethylene-glycol. We found that the surface modification improved the signal intensity for native MS of proteins and for CD-MS of adeno-associated viral capsids. Based on mechanistic comparisons, we hypothesize that the improvement is more likely due to an increased flow rate with coated ESI needles rather than less nonspecific adsorption. In any case, these surface-modified needles provide a simple and inexpensive method for improving the sensitivity of challenging analytes.12 month embargo; published: 19 May 2022This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Stratified analyses refine association between TLR7 rare variants and severe COVID-19

Summary: Despite extensive global research into genetic predisposition for severe COVID-19, knowledge on the role of rare host genetic variants and their relation to other risk factors remains limited. Here, 52 genes with prior etiological evidence were sequenced in 1,772 severe COVID-19 cases and 5,347 population-based controls from Spain/Italy. Rare deleterious TLR7 variants were present in 2.4% of young (<60 years) cases with no reported clinical risk factors (n = 378), compared to 0.24% of controls (odds ratio [OR] = 12.3, p = 1.27 × 10−10). Incorporation of the results of either functional assays or protein modeling led to a pronounced increase in effect size (ORmax = 46.5, p = 1.74 × 10−15). Association signals for the X-chromosomal gene TLR7 were also detected in the female-only subgroup, suggesting the existence of additional mechanisms beyond X-linked recessive inheritance in males. Additionally, supporting evidence was generated for a contribution to severe COVID-19 of the previously implicated genes IFNAR2, IFIH1, and TBK1. Our results refine the genetic contribution of rare TLR7 variants to severe COVID-19 and strengthen evidence for the etiological relevance of genes in the interferon signaling pathway