Native Mass Spectrometry Dissects the Structural Dynamics of an Allosteric Heterodimer of SARS-CoV-2 Nonstructural Proteins.

Structure-based drug design, which relies on precise understanding of the target protein and its interaction with the drug candidate, is dramatically expedited by advances in computational methods for candidate prediction. Yet, the accuracy needs to be improved with more structural data from high throughput experiments, which are challenging to generate, especially for dynamic and weak associations. Herein, we applied native mass spectrometry (native MS) to rapidly characterize ligand binding of an allosteric heterodimeric complex of SARS-CoV-2 nonstructural proteins (nsp) nsp10 and nsp16 (nsp10/16), a complex essential for virus survival in the host and thus a desirable drug target. Native MS showed that the dimer is in equilibrium with monomeric states in solution. Consistent with the literature, well characterized small cosubstrate, RNA substrate, and product bind with high specificity and affinity to the dimer but not the free monomers. Unsuccessfully designed ligands bind indiscriminately to all forms. Using neutral gas collision, the nsp16 monomer with bound cosubstrate can be released from the holo dimer complex, confirming the binding to nsp16 as revealed by the crystal structure. However, we observed an unusual migration of the endogenous zinc ions bound to nsp10 to nsp16 after collisional dissociation. The metal migration can be suppressed by using surface collision with reduced precursor charge states, which presumably resulted in minimal gas-phase structural rearrangement and highlighted the importance of complementary techniques. With minimal sample input (∼µg), native MS can rapidly detect ligand binding affinities and locations in dynamic multisubunit protein complexes, demonstrating the potential of an "all-in-one" native MS assay for rapid structural profiling of protein-to-AI-based compound systems to expedite drug discovery.

Investigating Daptomycin-Membrane Interactions Using Native MS and Fast Photochemical Oxidation of Peptides in Nanodiscs.

Daptomycin is a cyclic lipopeptide antibiotic that targets the lipid membrane of Gram-positive bacteria. Membrane fluidity and charge can affect daptomycin activity, but its mechanisms are poorly understood because it is challenging to study daptomycin interactions within lipid bilayers. Here, we combined native mass spectrometry (MS) and fast photochemical oxidation of peptides (FPOP) to study daptomycin-membrane interactions with different lipid bilayer nanodiscs. Native MS suggests that daptomycin incorporates randomly and does not prefer any specific oligomeric states when integrated into bilayers. FPOP reveals significant protection in most bilayer environments. Combining the native MS and FPOP results, we observed that stronger membrane interactions are formed with more rigid membranes, and pore formation may occur in more fluid membranes to expose daptomycin to FPOP oxidation. Electrophysiology measurements further supported the observation of polydisperse pore complexes from the MS data. Together, these results demonstrate the complementarity of native MS, FPOP, and membrane conductance experiments to shed light on how antibiotic peptides interact with and within lipid membranes.

Dissecting the structural heterogeneity of proteins by native mass spectrometry.

A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher-order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the "bottom-up" approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher-order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the "top-down" approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in-depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero-complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near-native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.

Investigating Antimicrobial Peptide-Membrane Interactions Using Fast Photochemical Oxidation of Peptides in Nanodiscs.

Antimicrobial peptides (AMPs) are an important part of the innate immune system and demonstrate promising applications in the fight against antibiotic-resistant infections due to their unique mechanism of targeting bacterial membranes. However, it is challenging to study the interactions of these peptides within lipid bilayers, making it difficult to understand their mechanisms of toxicity and selectivity. Here, we used fast photochemical oxidation of peptides, an irreversible footprinting technique that labels solvent accessible residues, and native charge detection-mass spectrometry to study AMP-lipid interactions with different lipid bilayer nanodiscs. We observed differences in the oxidation of two peptides, indolicidin and LL-37, in three distinct lipid environments, which reveal their affinity for lipid bilayers. Our findings suggest that indolicidin interacts with lipid head groups via a simple charge-driven mechanism, but LL-37 is more specific for Escherichia coli nanodiscs. These results provide complementary information on the potential modes of action and lipid selectivity of AMPs.

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes.

A growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological functions via lipid interactions extends to the diverse group of proteins that associate only peripherally with the lipid bilayer. However, the structural basis of these interactions remains challenging to study due to their transient and promiscuous nature. Recently, native mass spectrometry has come into focus as a new tool to investigate lipid interactions in membrane proteins. Here, we outline how the native MS strategies developed for integral membrane proteins can be applied to generate insights into the structure and function of peripheral membrane proteins. Specifically, native MS studies of proteins in complex with detergent-solubilized lipids, bound to lipid nanodiscs, and released from native-like lipid vesicles all shed new light on the role of lipid interactions. The unique ability of native MS to capture and interrogate protein-protein, protein-ligand, and protein-lipid interactions opens exciting new avenues for the study of peripheral membrane protein biology.

Expanding the Types of Lipids Amenable to Native Mass Spectrometry of Lipoprotein Complexes.

Native mass spectrometry (MS) has become an important tool for the analysis of membrane proteins. Although detergent micelles are the most commonly used method for solubilizing membrane proteins for native MS, nanoscale lipoprotein complexes such as nanodiscs are emerging as a promising complementary approach because they solubilize membrane proteins in a lipid bilayer environment. However, prior native MS studies of intact nanodiscs have employed only a limited set of phospholipids that are similar in mass. Here, we extend the range of lipids that are amenable to native MS of nanodiscs by combining lipids with masses that are simple integer multiples of each other. Although these lipid combinations create complex distributions, overlap between resonant peak series allows interpretation of nanodisc spectra containing glycolipids, sterols, and cardiolipin. We also investigate the gas-phase stability of nanodiscs with these new lipids towards collisional activation. We observe that negative ionization mode or charge reduction stabilizes nanodiscs and is essential to preserving labile lipids such as sterols. These new approaches to native MS of nanodiscs will enable future studies of membrane proteins embedded in model membranes that more accurately mimic natural bilayers. Graphical Abstract.

MetaUniDec: High-Throughput Deconvolution of Native Mass Spectra.

The expansion of native mass spectrometry (MS) methods for both academic and industrial applications has created a substantial need for analysis of large native MS datasets. Existing software tools are poorly suited for high-throughput deconvolution of native electrospray mass spectra from intact proteins and protein complexes. The UniDec Bayesian deconvolution algorithm is uniquely well suited for high-throughput analysis due to its speed and robustness but was previously tailored towards individual spectra. Here, we optimized UniDec for deconvolution, analysis, and visualization of large data sets. This new module, MetaUniDec, centers around a hierarchical data format 5 (HDF5) format for storing datasets that significantly improves speed, portability, and file size. It also includes code optimizations to improve speed and a new graphical user interface for visualization, interaction, and analysis of data. To demonstrate the utility of MetaUniDec, we applied the software to analyze automated collision voltage ramps with a small bacterial heme protein and large lipoprotein nanodiscs. Upon increasing collisional activation, bacterial heme-nitric oxide/oxygen binding (H-NOX) protein shows a discrete loss of bound heme, and nanodiscs show a continuous loss of lipids and charge. By using MetaUniDec to track changes in peak area or mass as a function of collision voltage, we explore the energetic profile of collisional activation in an ultra-high mass range Orbitrap mass spectrometer. Graphical abstract ᅟ.

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.

Engineering Nanodisc Scaffold Proteins for Native Mass Spectrometry.

Lipoprotein nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.