Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC (~70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC (~70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

Melanocortin 2 receptor (MC2R) is a G protein-coupled receptor (GPCR) for adrenocorticotropic hormone (ACTH), and its trafficking and signalling are associated with the melanocortin receptor accessory protein (MRAP). Mutations in either MC2R or MRAP disrupt this signalling and cause familial glucocorticoid deficiency. Here, we combine native mass spectrometry (MS) and hydrogen deuterium exchange mass spectrometry (HDX MS) to uncover how MRAP association and post-translational modification status shape the conformations of MC2R. Using native MS, we demonstrate that MC2R associates with MRAP or when MRAP is depleted the protein is extensively palmitoylated at the C-terminus. ACTH binding is restricted to the MC2R MRAP complex. By contrast antagonists shift the equilibrium toward MRAP-independent receptor populations. Our HDX MS analysis shows that ACTH binding induces global stabilisation of MC2R and the MRAP N terminus, consistent with reinforcement of the receptor accessory protein interface. Antagonist binding by contrast destabilises this interface and increases dynamics in transmembrane helix 2 (TM2). Notably, TM2 destabilisation is retained when the MRAP complex is depleted. Together, MRAP association and palmitoylation define distinct MC2R assemblies with ligand-dependent dynamics, suggesting new ways to influence MC2R pharmacology.

Intracellular lipid transport in eukaryotes is largely mediated by lipid transfer proteins (LTPs). Transport kinetics differ markedly among lipid species, implying selective lipid recognition by the involved proteins. Here, we characterize endogenous ligands of the human phospholipid transporters STARD2, STARD7, and STARD10 by multistage native mass spectrometry (MS). Our results demonstrate that they exhibit distinct lipid selectivities, with STARD7 binding a broad range of phospholipids, whereas STARD2 and STARD10 preferentially copurify with poly- and di-unsaturated phospholipids, respectively. We link this acyl chain selectivity to tissue-specific LTP expression patterns and show that LTP expression levels modulate lipid metabolism. Through site-directed mutagenesis and molecular dynamics simulations, we further identify a conserved arginine that is essential for phospholipid binding in STARD7 but dispensable in STARD2 and STARD10. To investigate regulation of LTP activity, we mapped phosphorylation sites by native top-down MS and found that STARD2 and STARD10 are phosphorylated in membrane-binding regions. Liposome-based assays revealed that phosphorylation abolishes lipid transfer activity of STARD10 and that lipid selectivity influences the transfer rates of different lipid probes. Together, our results demonstrate that LTPs exhibit distinct lipid binding preferences and suggest that cells finely tune lipid homeostasis by regulating LTP expression levels and activity. Lipid transfer proteins organize intracellular lipid distribution. Here, the authors show that seemingly redundant phospholipid transfer proteins have distinct acyl chain preferences, and suggest that their tissue-specific expression levels and phosphorylation fine-tune membrane composition.

Abstract Sodium-proton exchangers (NHEs) are found in all cells to regulate intracellular pH, sodium levels and cell volume. In humans, there are nine different NHE transporters (SLC9A1-9), which vary in tissue distribution, kinetics and regulation. NHE6 localizes to endosomal membranes and mutations in the protein are known to cause the X-linked neurological disorder Christianson syndrome. Despite its importance, the structural basis of NHE6 function and regulation is unclear. Here we report four cryo-electron microscopy structures of rat NHE6 between 2.2 and 3.3 Å resolution, revealing its homodimeric structure, ion binding and remodelling by lipids. We characterize a lipid-binding site between the protomers that accommodates the endosomal-specific phosphatidylinositol 3-phosphate (PI3P) lipid. Using solid-supported membrane (SSM)-based electrophysiology we demonstrate that NHE6 transports both Na + and K + ions and that PI3P enhances NHE6 stability and activity. Furthermore, we identify a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) lipid, which interacts with the C-terminal domain of NHE6 to stabilize an auto-inhibited state. We further demonstrate that NHE6 is non-functional when mislocalized to the plasma membrane where PI(4,5)P 2 is primarily located. We propose the lipid-dependent regulation has evolved to shut-down NHE6 activity during recycling of endosomes at the plasma membrane.

Point-of-care diagnostic tools, such as lateral flow assays (LFAs), play a critical role in disease management and outbreak control. LFAs detect the presence of target antigens in disease-relevant biofluids, utilizing nanoparticles (termed detection probes) to produce colorimetric readouts. However, significant intra- and interpatient variation in the biochemical composition of biofluids has downstream consequences for assay performance. Robust LFAs must be able to function alongside such variability to produce reliable and reproducible test outcomes. Beyond this, biofluids (such as serum) contain significant amounts of proteins, which can interact with detection probes used in LFAs to form a protein corona. The consequences of protein corona formation on LFA performance are poorly understood. Using a model antigen-biofluid LFA (human epidermal growth factor receptor 2 (HER2) and human serum), we observed significant discrepancies in LFA performance when using conventional nanoparticle functionalization methods, including the use of generic, nonhuman protein blocking agents. To overcome these performance differences, we developed a methodology for Bionano interface Optimization for LFA Design (termed BOLD). The BOLD workflow employs mass spectrometry-based proteomics to characterize the native protein corona, followed by formation of an engineered corona to produce an optimized bionano interface. We identified a specific protein (kininogen-1, KNG1) that demonstrated negative interference, significantly reducing the observed LFA test line intensity. This experimental finding is complemented by Molecular Dynamics simulations, which probe the binding modes of KNG1 to platinum nanoparticles. Further, through the employment of an apolipoprotein engineered corona (apolipoprotein A1, B, and C3), a robust LFA was developed, increasing test line intensity and significantly reducing intersample variation (with over a 4-fold improvement in the coefficient of variation). Overall, the BOLD workflow presents a method for the rational optimization of detection probes in LFAs through the characterization of the bionano interface to produce robust LFAs.

Covalent ligands are widely used to label, probe, and modulate proteins, but peptide-centric readouts obscure how modifications colocalize on intact proteoforms. This can limit insight into ligand mechanism, modification stoichiometry, and the architecture of multisite protein conjugates. We present a general native top-down mass spectrometry workflow that quantifies electrophile reactivity directly on intact proteins. Using NHS esters as a model electrophile class, we apply a deconvolution framework to infer differential reactivity at primary amines across promiscuous, multisite modification patterns. The approach preserves full modification connectivity, avoids sample-preparation artifacts associated with denaturation and digestion, and should extend to electrophiles with unknown reactivity. Overall, this framework provides a general platform for designing covalent therapeutics, bioconjugates, and activity-based probes with proteoform-level resolution.

Bifunctional diazirine lipids are versatile tools for mapping protein-lipid interactions and cellular localization by photo-cross-linking. Yet, the cross-linking efficiency of these probes has not been systematically evaluated. We use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-cross-linking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein-lipid cross-links, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows.

Gram-positive bacteria protect their cell envelope through d-alanylation of lipoteichoic acid (LTA), a process initiated by DltA-mediated activation of d-alanine and loading onto the carrier protein DltC. Although structural and biochemical studies have established key features of DltA catalysis, direct observation of adenylate formation and carrier protein loading within a reconstituted DltA-DltC system has remained limited. Here, we reconstituted the Bacillus subtilisd-alanylation pathway and used native mass spectrometry to resolve DltA-dependent reaction intermediates and products. We detected ATP-dependent adenylation of d-alanine by DltA followed by transfer to holo-DltC. This process was inhibited by a sulfamoyl-adenosine compound that mimics the adenylate intermediate. Mutational analysis of the conserved DltA P-loop revealed position-specific effects on catalysis, highlighting structural features that govern substrate processing. Together, these findings define regulatory steps in the Dlt pathway and identify opportunities for targeted inhibition. The same strategy should be adaptable to other systems in which transient acyl- or aminoacyl-carrier protein intermediates are difficult to monitor directly.

Abstract Membrane lipids are central regulators of G protein–coupled receptor (GPCR) function. Defining receptor-specific lipid interactions in native, fully modified mammalian systems remains challenging. Extensive post-translational modifications generate heterogeneous proteoforms that confound conventional mass spectrometry approaches. Here we introduce REVEAL (REceptor enVironment Elucidation by Activated Lipid-release), an automated native top-down mass spectrometry strategy that discriminates specifically bound lipids from background. Applied here to intact, heterogeneous mammalian membrane protein complexes, incubated with a brain polar lipid extract (>1000 components), we define receptor-specific lipid-binding for two neuronal class C GPCRs. We show that agonism remodels lipid occupancy, selectively enriching a reduced repertoire of bound lipids. Plasmalogen lipids emerge as persistent binders across all conformational states of the metabotropic glycine receptor and are preferentially depleted under oxidative stress, implying a protective role at the receptor surface. These findings position lipids as dynamic regulators of both function and response to the cellular redox environment.

Native MS offers a clear picture of membrane protein stoichiometry and interactions, but it lacks direct structural insights at high resolution. Here, we examine the extent to which solution-phase structure and architecture can be retained after native, soft-landing electrospray ion beam deposition (ESIBD) by interrogating several membrane-protein complexes of diverse folds and oligomeric states by cryoEM. The overall protein architectures with secondary structure motifs can be observed after gas-phase transfer, soft landing, and embedding in amorphous ice. Notably, we determined the structure of the ammonium transporter AmtB at sub-3 Å resolution. It is nearly identical to the structure of the plunge-frozen control and even shows an extended C-terminal segment of AmtB, a dynamic region absent in the solution-phase structure. Our analysis shows that detergent adducts preserve membrane protein structure in vacuum by minimising destabilization of solvent-exposed regions and stabilization through additional polar contacts in vacuo. Molecular dynamics (MD) simulations support these results, suggesting that a monolayer shell of surfactant adducts avoids destabilization driven by unshielded polar residues and disruption of hydrogen bond networks. Overall, our findings provide a structural framework for integrating native MS with cryo-EM showing that gas-phase transfer and surfactant stabilisation preserves key architectural features and high-resolution structure of membrane proteins. ### Competing Interest Statement T.E. is an employee of Thermo Fisher Scientific, manufacturer of Q Exactive UHMR, Aquilos, Arctica and Krios instruments used in this research. C.V.R. is a cofounder and consultant of OMass Therapeutics. T.E. and S.R. have applied for related patents (US2023028024, GB2614323). All other authors declare no competing interests. UK Research and Innovation, EP/Z001684/1, MR/V028839/1, EP/V051474/1, BB/V019694/1 Wellcome Trust, 228310/Z/23/Z, 221795/Z/20/Z, 218482/Z/19/Z Swedish Research Council, 2020-04825

As health needs in our society evolve, the field of drug discovery must undergo constant innovation and improvement to identify novel targets and drug candidates. Owing to its ability to simultaneously capture biological interactions and provide in-depth molecular characterisation of the species involved, native mass spectrometry is starting to play an important role in this endeavour. Here, we discuss recent contributions that native mass spectrometry has made to drug discovery including deciphering protein-small molecule interactions, unravelling biochemical pathways, and integrating with complementary structural approaches.

Membrane proteins and lipids are essential for a wide range of cellular processes, making their structural characterisation essential for understanding biological function. However, the amphipathic nature of membrane proteins poses a significant challenge for traditional structural biology techniques. Membrane mimetics offer an alternative approach to studying membrane proteins in more native-like environments. Among them, peptidiscs have emerged as a promising tool for stabilising membrane proteins, allowing reconstitution from detergent micelles into a detergent-free, native-like environment that preserves structural integrity. While peptidiscs have shown utility in techniques such as mass photometry and cryo-EM, their compatibility with native mass spectrometry (MS) remains largely unexplored. In this study, we evaluate the feasibility of using peptidiscs for native MS analysis of membrane proteins and their complexes, focusing on the antibiotic resistance efflux pump AceI and the β-barrel assembly machinery (BAM complex). We reconstituted these proteins into peptidiscs using both 'on-column' and 'on-bead' assembly methods and assessed complex integrity and stability post-reconstitution using native MS. Our findings highlight the potential of peptidiscs as a tool for native MS-based structural characterisation of membrane protein and their assemblies.

Understanding the dynamics of membrane protein–ligand interactions within a native lipid bilayer is a major goal for drug discovery. Typically, cell-based assays are used, however, they are often blind to the effects of protein modifications. In this study, using the archetypal G protein-coupled receptor rhodopsin, we found that the receptor and its effectors can be released directly from retina rod disc membranes using infrared irradiation in a mass spectrometer. Subsequent isolation and dissociation by infrared multiphoton dissociation enabled the sequencing of individual retina proteoforms. Specifically, we categorized distinct proteoforms of rhodopsin, localized labile palmitoylations, discovered a Gβγ proteoform that abolishes membrane association and defined lipid modifications on G proteins that influence their assembly. Given reports of undesirable side-effects involving vision, we characterized the off-target drug binding of two phosphodiesterase 5 inhibitors, vardenafil and sildenafil, to the retina rod phosphodiesterase 6 (PDE6). The results demonstrate differential off-target reactivity with PDE6 and an interaction preference for lipidated proteoforms of G proteins. In summary, this study highlights the opportunities for probing proteoform–ligand interactions within natural membrane environments. G protein-coupled receptors and their effectors can now be released directly from a lipid bilayer using infrared irradiation for proteoform-level characterization by native top-down mass spectrometry. This represents a critical development for drug discovery, as the direct role of post-translational modifications in protein–protein and protein–drug interactions can be characterized.

In all living organisms, membrane proteins play a crucial role in governing essential biological functions, such as cellular signaling and molecular transport. These functions rely on intricate interactions with a variety of biomolecules, including substrates, proteins, metabolites, and lipids. Any disruption or alteration to these interactions can result in disease. Therefore, comprehending the complex assemblies of membrane proteins, and their intrinsic interactions, is crucial for unraveling the mechanisms of cellular regulation and has implications in disease pathology. Over the past three decades, native mass spectrometry (MS) has emerged as a pivotal tool for investigating the structure and dynamics of proteins, including membrane protein complexes. In this review, we discuss recent developments in instrumentation that advance our ability to characterize membrane proteins in their native context. As we transition toward increasingly complex eukaryotic systems, we show how this information is translated into an understanding of disease. We also highlight preliminary studies in which native MS has been used to sequence and localize membrane protein complexes within endogenous tissue. This level of detail offers the promise of informing about the molecular mechanisms of disease states.

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

Protein-lipid interactions are critical for maintaining membrane protein structure and regulating diverse protein functions. Native mass spectrometry (MS) has emerged as a powerful technique for the direct observation and characterization of protein-lipid complexes. However, intact mass measurements alone cannot resolve important structural details such as the identity of lipid acyl chains and their modifications. To fully characterize protein-bound lipids, we present a multistage native MS method that leverages ultraviolet photodissociation to elucidate the precise molecular composition of heterogeneous protein-lipid assemblies. We demonstrate the utility of this approach for both soluble and membrane proteins. First, we comprehensively define the endogenous lipids bound to the bacterial transporter MlaC, distinguishing between unsaturated and cyclopropane lipids, and localizing acyl chains and their modifications. Next, we characterize and quantify phospholipids associated with the bacterial membrane protein AqpZ and show that the approach can be extended to more complex cardiolipins containing four lipid chains. Together, our workflow provides detailed structural insights into protein-lipid interactions and offers a path toward uncovering protein-specific metabolic regulation that is not accessible through classical lipidomics workflows.

The maintenance of lipid asymmetry (Mla) system in gram-negative bacteria transfers phospholipids between the outer and inner membrane to maintain the outer membrane asymmetry. Misplaced phospholipids are extracted from the outer leaflet of the outer membrane by MlaA, transferred to the periplasmic lipid transporter MlaC, and shuttled to the inner membrane. We set out to investigate the lipid transfer between MlaA and MlaC using native mass spectrometry, with the aim of determining the lipid preferences of MlaC and whether MlaA preselected lipids for MlaC. First, we characterized the lipids that copurified with overexpressed MlaC, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), and following delipidation noted a headgroup-independent enrichment of cyclopropane lipids. Under native expression conditions, we found that PG is three-fold enriched on MlaC compared to its abundance in the membrane. Next, we isolated and characterized OmpF 3 –MlaA complexes and demonstrated their ability to enhance loading of delipidated MlaC with bacterial and nonbacterial phospholipids. We then captured the intact ternary lipid shuttle (OmpF 3 –MlaA–MlaC) and demonstrated that PG dissociates this transient complex, releasing lipid-bound MlaC. Together our results point to a high population of endogenous PG on periplasmic MlaC, which likely arises from disassembly of the lipid shuttle to maintain lipid asymmetry for cell viability.

Protein modifications drive dynamic cellular processes by modulating biomolecular interactions, yet capturing these modifications within their native structural context remains a significant challenge. Native top-down mass spectrometry promises to preserve the critical link between modifications and interactions. However, current methods often fail to detect uncharacterized or low-abundance modifications, limiting insights into proteoform diversity. To address this gap, we introduce precise and accurate Identification Of Native proteoforms (precisION), an interactive end-to-end software package that leverages a robust, data-driven fragment-level open search to detect, localize and quantify 'hidden' modifications within intact protein complexes. Applying precisION to four therapeutically relevant targets-PDE6, ACE2, osteopontin (SPP1) and a GABA transporter (GAT1)-we discover undocumented phosphorylation, glycosylation and lipidation, and resolve previously uninterpretable density in an electron cryo-microscopy map of GAT1. As an open-source software package, precisION offers an intuitive means for interpreting complex protein fragmentation data. This tool will empower the community to unlock the potential of native top-down mass spectrometry, advancing integrative structural biology, molecular pathology and drug development.

The alarming rise in superbugs that are resistant to drugs of last resort, including vancomycin-resistant enterococci and staphylococci, has become a significant global health hazard. Here we report the click chemistry synthesis of an unprecedented class of shapeshifting vancomycin dimers (SVDs) that display potent activity against bacteria that are resistant to the parent drug, including the ESKAPE pathogens, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA) as well as vancomycin-resistant S. aureus (VRSA). The shapeshifting modality of the dimers is powered by a click-linked bullvalene core, hence exploiting the dynamic covalent rearrangements of the fluxional carbon cage and creating ligands with the capacity to inhibit bacterial cell wall biosynthesis. The new shapeshifting antibiotics are not disadvantaged by the common mechanism of vancomycin resistance resulting from the alteration of the C-terminal dipeptide with the corresponding D-Ala-D-Lac depsipeptide. Further, evidence suggests that the shapeshifting ligands destabilize the complex formed between the flippase MurJ and lipid II, inferring the potential for a new mode of action for polyvalent glycopeptides. The SVDs show little propensity for acquired resistance by enterococci, suggesting that this new class of shapeshifting antibiotic will display durable antimicrobial activity not prone to rapidly acquired clinical resistance.