Lipids and EGCG 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, studying the intersection of protein complex formation, membrane interactions, and bilayer disruption simultaneously is challenging. 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 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipids but interacted strongly with anionic 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) lipids, eventually leading to membrane disruption. Unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (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 fibrillization, it does not inhibit α-synuclein from associating with the membrane.

Stability and Dissociation of Adeno-Associated Viral Capsids by Variable Temperature-Charge Detection-Mass Spectrometry.

Adeno-associated viral (AAV) vectors have emerged as gene therapy and vaccine delivery systems. Differential scanning fluorimetry or differential scanning calorimetry is commonly used to measure the thermal stability of AAVs, but these global methods are unable to distinguish the stabilities of different AAV subpopulations in the same sample. To address this challenge, we combined charge detection-mass spectrometry (CD-MS) with a variable temperature (VT) electrospray source that controls the temperature of the solution prior to electrospray. Using VT-CD-MS, we measured the thermal stabilities of empty and filled capsids. We found that filled AAVs ejected their cargo first and formed intermediate empty capsids before completely dissociating. Finally, we observed that pH stress caused a major decrease in thermal stability. This new approach better characterizes the thermal dissociation of AAVs, providing the simultaneous measurement of the stabilities and dissociation pathways of different subpopulations.

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.

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 the 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 deconvolution 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 the 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.

Chemical Additives Enable Native Mass Spectrometry Measurement of Membrane Protein Oligomeric State within Intact Nanodiscs.

Membrane proteins play critical biochemical roles but remain challenging to study. Recently, native or nondenaturing mass spectrometry (MS) has made great strides in characterizing membrane protein interactions. However, conventional native MS relies on detergent micelles, which may disrupt natural interactions. Lipoprotein nanodiscs provide a platform to present membrane proteins for native MS within a lipid bilayer environment, but previous native MS of membrane proteins in nanodiscs has been limited by the intermediate stability of nanodiscs. It is difficult to eject membrane proteins from nanodiscs for native MS but also difficult to retain intact nanodisc complexes with membrane proteins inside. Here, we employed chemical reagents that modulate the charge acquired during electrospray ionization (ESI). By modulating ESI conditions, we could either eject the membrane protein complex with few bound lipids or capture the intact membrane protein nanodisc complex-allowing measurement of the membrane protein oligomeric state within an intact lipid bilayer environment. The dramatic differences in the stability of nanodiscs under different ESI conditions opens new applications for native MS of nanodiscs.

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.