Angewandte Chemie

Protein synthesis supports most biological processes. In the brain in particular, protein synthesis plays a critical role in physiological and pathological states. Here, we describe Tellurophene-Alkyne Cycloaddition-mediated Amino acid Tagging (TeACAT), a versatile strategy for fast, facile, and flexible tagging of newly synthesized proteins in mice. TeACAT is based on metabolic incorporation of the non-canonical amino acid TePhe into proteins by the endogenous protein synthesis machinery. Due to their high similarity, TePhe can efficiently replace canonical Phe without dietary or genetic manipulation. The subsequent bio-orthogonal reaction of TePhe with either fluorescent dyes or affinity handles enables both visualization and affinity enrichment of proteins synthesized during TePhe exposure. TeACAT is compatible with immunofluorescence for cell-type specific visualization of protein synthesis with subcellular resolution and can be used in conjunction with routine proteomics to identify and quantify newly synthesized proteins. Robust incorporation into the mouse proteome was observed on the scale of hours to days, allowing the interrogation of various biological processes. In summary, TeACAT enables the visualization and quantification of protein synthesis with minimal perturbation for biological discoveries.

Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.

Membrane proteins are central to transport, signaling, and pharmacological regulation, yet their direct functional reconstitution into defined membrane environments remains technically challenging. Detergent-based workflows have enabled major advances in membrane protein research, but some applications require complementary strategies that better preserve native-like lipid environments. Cell-based expression approaches, meanwhile, require long incubation times and suffer from cell-type-dependent variability. Here, we establish nanodiscs as modular carriers for the rapid delivery of both lipids and full-length membrane proteins into model and cellular membranes. Using supported lipid bilayers, we first show that membrane scaffold protein (MSP) nanodiscs mediate efficient lipid transfer within minutes, with fluorescence recovery after photobleaching confirming lateral mobility of the delivered lipids. We then extend this strategy to the bacterial calcium channel BsYetJ, achieving concentration-dependent protein incorporation and single-molecule diffusion within supported lipid bilayers. Importantly, BsYetJ-loaded nanodiscs enable direct reconstitution of functional channels into intact mammalian plasma membranes across multiple cell lines. Calcium imaging demonstrates robust BsYetJ-mediated calcium influx, confirming that the delivered channel retains ion-conductive activity after transfer into heterologous cellular membranes. Crucially, this nanodisc-mediated delivery bypasses the variable trafficking pathways inherent to different host systems, allowing for the direct reconstitution of membrane proteins into target membranes while preserving their functional activity. Furthermore, unlike MSP nanodiscs, styrene-maleic acid (SMA) nanodiscs can directly capture membrane proteins from native cell membranes. This capability makes them particularly well-suited for studying complex and challenging membrane proteins. Therefore, we further generalize this platform using SMA nanodiscs . We demonstrate that, similar to MSP nanodiscs, SMA nanodiscs can efficiently deliver lipid cargo to supported bilayers and mammalian cells. By directly capturing full-length dopamine D2 receptor from cellular membranes and transferring it into naive target cells, we achieve functional GPCR reconstitution, as validated by specific binding of a custom fluorescent agonist. Together, these results demonstrate that nanodiscs can serve not only as stabilizing membrane mimetics but also as active delivery vehicles for on-demand membrane protein reconstitution. This approach provides a rapid and broadly applicable platform for interrogating ion channels, GPCRs, and other challenging pharmacological targets in user-defined membrane environments.

The initial attachment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to host cell sialosides is critical for infection, yet its precise receptor specificity remains poorly understood. Here, we describe a chemoenzymatic methodology to synthesize a comprehensive panel of 6-sulfo sialyl Lewisx (6-sulfo-SLex) containing glycans. Our approach entails the enzymatic assembly of an oligo-lactosamine chain modified at specific positions with N-trifluoroacetyl-glucosamine (GlcNTFA) moieties. Mild base treatment removes the TFA group to yield glucosamine, which effectively blocks enzymatic fucosylation. By leveraging this approach alongside the unique substrate selectivity of GlcNAc-6-O-sulfotransferases 2 (CHST-2), we achieved the selective preparation of fucosylated 6-sulfo-SLex glycans. Microarray screening of these printed glycans revealed that a 6-sulfo-SLex derivative presented on an extended LacNAc chain is the preferred host receptor for MERS-CoV. Conjugation of this lead compound to a polyglycerol-based dendrimer generated a multivalent inhibitor that potently blocks hemagglutination of human red blood cells by the MERS-CoV spike protein N-terminal domain (NTD). Furthermore, computational modeling demonstrated that the fucose moiety does not directly contact the viral spike protein. Instead, it pre-organizes the ligand into a favorable conformation, enabling a critical salt bridge between the glycans sulfate group and the guanidinium side chain of viral residue Arg307.

The BCL-2 family proteins are key regulators of apoptosis, functionally divided in pro- and anti-apoptotic proteins, with a third group acting as regulators. Their ability to partition between the cytosol and intra-cellular membranes (essentially the mitochondrial outer membrane) is a primary regulator of their functions. A second contributor is their ability to form homotypic complexes (pro-pro or anti-anti) or heterotypic complexes (pro-anti). If the structures of monomeric cytosolic members have largely been characterized, the functional and structural study of membrane-embedded proteins remains incomplete. Unlocking this knowledge is expected to enable evaluating new therapeutic strategies to either activate pro-apoptotic members, or inactivate anti-apoptotic ones. Lipid bilayer nanodiscs and improved cell-free protein synthesis have provided the technical breakthrough to achieve the description at the atomic level of conformations and higher order assemblies of these proteins in their membrane-associated states. Here we describe detailed and straightforward protocols for generating nanodisc-inserted members of the Bcl-2 family, through the example of anti-apoptotic Bcl-xL, and pro-apoptotic Bax and Bak. Full-length, untagged proteins are expressed from bacterial extracts in the presence of pre-assembled nanodiscs to allow co/post-translational insertion in lipid bilayer, followed by affinity chromatography purification. A more detailed characterization is presented for Bak, to exemplify structural and mechanistic studies enabled by these methods. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/745005v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@5da1d1org.highwire.dtl.DTLVardef@12aca96org.highwire.dtl.DTLVardef@5a3e73org.highwire.dtl.DTLVardef@ba009d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Weak, reversible interactions underpin biomolecular recognition, and single-molecule fluorescence (smF) imaging techniques can provide unprecedented insight into those biological processes. Unfortunately, such studies often require micromolar concentrations of fluorophore-labeled biomolecules, which is beyond the accessible range of conventional smF microscopies. Here, we describe a surface-functionalization method based on cloud-point polyethylene glycol (PEG) grafting that enables widefield smF microscopy measurements at micromolar concentrations without the use of nanophotonic devices. Using conventional total internal reflection fluorescence (TIRF) microscopy, we detected single-molecule fluorescence resonance energy transfer (smFRET) from surface-tethered, donor-labeled target molecules with up to 8 micromolar concentrations of freely diffusing, acceptor-labeled analyte molecules in the background--two orders of magnitude higher than typical studies in the literature. Weak, DNA-hybridization and protein-RNA binding equilibria were measured across micromolar range titrations. Together with advances in high-background data analysis, the robust method presented here enables kinetic and thermodynamic analyses of weak biomolecular interactions, especially those limited by nonspecific adsorption and high fluorescence backgrounds, using only standard smF instrumentation.

Glycosaminoglycan (GAG) polysaccharides play vital roles in animal physiology and disease1. Their diverse and intricate patterns of sulfation and epimerization endow them with an extensive potential to encode functional information2,3. GAG characterization, however, remains a formidable challenge for state-of-the-art ensemble-based techniques4,5. Single-molecule techniques are uniquely suited for analysing complex mixtures6,7. Here, we report the single-molecule resolution and counting of diverse GAG di- and oligosaccharides derived from longer heterogeneous chains as part of a deconvolutive nanopore-based workflow that requires no fractionation and is operationally simple. Modular chemical deacylation and amino-selective ring-contractive formation of electrophilic aldehydes enable the parsing of libraries of GAG structures into simplified sets of reactive anhydrosugars for nanopore readout via reversible covalent adduct formation. Discrete clustering of event amplitudes enables direct sugar sizing ([~]10 % step change per residue), which can be coupled to precisely resolved amplitude differences that further reveal sugar fine structure--including the number and position of sulfate groups ([~]2 % step change per sulfate) alongside single- atom stereochemistry ([~]0.5 % step change between epimers). Guided by chemical logic, the reverse mapping of resolved anhydrosugars to their precursors covers [~]84-100 % of all disaccharides and their eliminative digestion variants in natural heparan sulfate (HS). We demonstrate the practical utility and scope of our approach through the compositional analysis of a panel of HS polysaccharides that together encompass natural GAG structural diversity. Moreover, we detect contaminants in heparin, including oversulfated chondroitin sulfate found in an authentic pharmaceutical heparin sample previously implicated in a global healthcare crisis. Together, our results suggest a general chemo-biophysical framework for the precise and sensitive characterization of GAGs that extends to other aminosugar biopolymers. When adapted for portable, widely used nanopore sequencing devices, our approach may offer a path towards the long-sought democratization of glycan analysis.

Investigating the conformational dynamics of intrinsically disordered proteins (IDPs) is essential to understanding how their structural heterogeneity underlies function and how their dysregulation contributes to diseases. Here, we utilized an MspA nanopore-based approach for studying the conformational dynamics and interactions of IDPs at the single-molecule level. The platform was demonstrated using the intrinsically disordered transactivation domain of tumor suppressor p53 (p53-TAD), one of the important proteins in cancer biology. We showed that MspA can stably capture p53-TAD and resolve up to six distinct current states with frequent interconversions, revealing a rich conformational landscape. The nanopore also detected the effect of a cancer-associated double mutational variant, N29K/N30D. Combining experiments with steered molecular dynamics simulations, we showed that the mutant sampled compact conformational states more frequently than wild type, consistent with previous NMR studies. Importantly, the MspA platform enabled direct monitoring of E3 ligase MDM2 binding to p53-TAD and resolved how this interaction is inhibited by anti-cancer compound epigallocatechin gallate (EGCG). Notably, EGCG stabilizes one of the six states sampled by p53-TAD, providing a mechanistic explanation for its inhibitory effect. Together, these findings demonstrate the promise of the nanopore platform for label-free monitoring of IDP conformational dynamics, modulation, binding and inhibition at single-molecule resolution.

Chemical proteomics can provide global portraits of small molecule-protein interactions in native biological systems. Such ligandability maps have, however, been mostly restricted to readily accessible cell lines and primary immune cells. Here, we describe an activity-based protein profiling (ABPP) strategy for mapping the covalent ligandability of primary brain cells isolated from mice. By investigating sets of stereochemically defined electrophilic small molecules (stereoprobes), we identify liganding events for diverse brain cell proteins, including many with nervous system-enriched expression. In this category were multiple hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, which we show are covalently liganded by tryptoline acrylamide stereoprobes at a conserved cysteine in their cyclic nucleotide-binding domain. The stereoprobes were found to block cAMP-dependent shifts in voltage dependence while sparing basal activity of HCN channels. We thus describe an advanced ABPP platform for identifying ligands targeting nervous system-enriched proteins, including chemical probes that modulate HCN channel function in cells.

Small-molecule inhibitors rely on molecular recognition within suitable binding pockets, leaving many disease-associated proteins difficult to target. Here, we introduce the concept of a single-atom inhibitor in which gold (Au) engages critical cysteine residues of oncogenic drivers to suppress their activity. We used an AI-assisted few-shot learning approach to identify EGFR-targeting peptides for in vivo Au delivery and showed that the lead candidate, 10714, promoted Au accumulation in EGFR-expressing cells and tumors. In vivo, Au exploited its intrinsic affinity for cysteine to inhibit two structurally distinct oncogenic proteins, engaging Cys797 in EGFR T790M and the mutation-derived Cys12 in KRAS G12C adjacent to their respective nucleotide-binding pockets. Structural and computational analyses supported stabilization of inactive nucleotide-bound states, while mutation of these cysteine residues abrogated Au-mediated inhibition. 10714-Au consequently suppressed oncogenic signaling, reduced non-small-cell lung cancer cell viability, and inhibited tumor growth in EGFR- and KRAS-mutant xenograft models and patient-derived organoids. These findings establish proof of principle for single-atom inhibition across structurally distinct oncogenic drivers and suggest that localized atomic coordination could provide an alternative mode of target engagement to conventional pocket-dependent inhibition.

Understanding the physicochemical factors that govern siRNA nanocarrier assembly is essential for the rational design of effective delivery systems. By optimizing various lipid compositions, cholesterol content and PEG length we created a peptide-functionalized cationic liposomal platform made of DOPE/TAP lipids with cholesterol-anchored nona-arginine (R9-Chol) for siRNA complexation, intracellular transport and effective silencing of the target EGFR gene. Analysis of {zeta}-potential and dynamic light scattering allowed to rationally design formulation of stable, monodisperse nanoscale lipoplexes with a positive surface charge. With fluorescence polarization, circular dichroism and agarose gel electrophoresis we found an optimal siRNA:liposome complexation ratio of 1:77, which protected siRNA from ribonuclease-mediated degradation. Morphological imaging confirmed a shift from discrete vesicular structures to organized multilamellar lipoplexes, consistent with electrostatically driven self-assembly. In cellular studies, the optimized nanocarrier promoted efficient uptake of fluorescent siRNA in MDA-MB-231 cells and achieved functional delivery of anti-EGFR, leading to substantially reduced expression of the target gene at both transcript and protein levels. This work offers mechanistic understanding of peptide-assisted lipid:siRNA assembly and positions R9-functionalized DOPE/TAP liposomes as a promising platform for siRNA delivery.

Residual dipolar couplings (RDCs) obtained via magnetic field-induced alignment offer a powerful, media-free approach for the structural analysis of biomolecules. However, their detection in short, fast-tumbling nucleic acids remains elusive at conventional magnetic fields due to insufficient alignment and sensitivity. Here, we demonstrate the direct observation of these RDCs at 1.3 GHz in a 14-mer hairpin fragment derived from an HIV-1 Vif-targeting aptamer. The 1JNH scalar couplings of imino protons were measured at fields ranging from 600 MHz to 1.3 GHz. While the coupling constants remained invariant between 600 and 900 MHz, a clear deviation was exclusively captured at 1.3 GHz for all base-paired stem residues, demonstrating the first media-free detection of field-induced RDCs in a short RNA of this size. This breakthrough arises from a synergistic B07/2 scaling, combining enhanced alignment ({propto} B02) and sensitivity ({propto} B03/2). These RDCs showed excellent agreement with the NOE-derived structure. Additionally, the flexible loop residue G8 exhibited no detectable RDC, but displayed a field-dependent TROSY/anti-TROSY intensity inversion at 1.3 GHz, reflecting an unusual 1H chemical shift anisotropy (CSA) tensor that corroborates the local base-packing environment. Our findings highlight 1.3 GHz NMR as an indispensable tool for the structural analysis of short RNAs.

Multiplexed imaging of biomolecular networks in living cells is limited by the small number of spectral separable fluorophores and the need to monitor dynamic processes in real time. Here, we present logicFRIES, a fluorogenic RNA (FR)-based platform that enables eight-plex live-cell imaging through programmable, logic-gated activation coupled with sequential fluorescence imaging. By integrating small-molecule-binding RNA aptamers into dye-activating fluorogenic RNAs, we engineered trigger-responsive FR reporters. This design implements an AND-gated mechanism in which fluorescence activation requires both a cognate trigger molecule and its corresponding fluorogenic dye, thereby expanding multiplexing capacity without adding new fluorophores. Using three membrane-permeable triggers, tetracycline, ASP2905, and guanine, we generated three distinct trigger-defined activation states for each engineered Broccoli and Pepper FR. Combined with orthogonal Corn/DFHO and DNB/TMR-DN reporter pairs, logicFRIES supports eight-plex imaging through sequential trigger/dye addition, imaging, and wash-based stripping cycles. We demonstrate robust, specific, and reversible fluorescence switching of these multiplexed FR reporters in living HEK293T and SKBR3 cells. Overall, logicFRIES extends live-cell imaging beyond conventional spectral limitations and provides a modular foundation for potentially developing multiplexed sensors targeting endogenous RNAs, proteins, and small molecules in complex cellular systems.

Lipid metabolism is dynamically rewired across tissues in response to developmental, environmental and therapeutic cues. This adaptation drives treatment resistance in a range of human pathologies, but current lipidomic techniques fail to capture the underlying mechanisms, relying on steady-state measurements from homogenised samples that obscure spatial heterogeneity and pathway flux. Here we introduce spatial tracing of isotopically labelled lipids (STILL-13C), a workflow that uses stable isotope tracing and high-resolution mass spectrometry imaging (MSI) to map lipid metabolic flux directly in intact human tissues with unprecedented pathway coverage. STILL-13C overcomes longstanding limitations of bulk and MSI-based analyses by spatially resolving isotopologue labelling of simple and complex lipids, enabling simultaneous tracing of fatty acid synthesis, remodelling and multiple convergent pathways required for phospholipid assembly, while preserving tissue architecture and regional metabolic context. Applied to patient-derived prostate cancer explants cultured ex vivo, STILL-13C revealed heterogeneity in lipid pathway activity between neighbouring epithelial regions and spatially resolved responses to pathway inhibition. This work establishes a broadly applicable platform for investigating spatial heterogeneity in lipid metabolic flux and its perturbation in intact tissues.

Glycine is a vital extracellular signal in bacteria, plants, and the brain. Although RNA-based sensors detect glycine in cells, their extracellular application in native biological environments is limited by enzymatic degradation from nucleases. Mirror-image RNA is nuclease-resistant and preserves the tertiary structure required for RNA function, but synthesizing long L-RNAs such as the 170-nt glycine sensor (glyS) remains challenging. Here, we applied cross-chiral ligation with natural D-RNA ribozymes to assemble a mirror-image L-RNA glycine sensor (L-glyS). Optimization of the ligation conditions enabled up to 68% conversion to the full-length sensor. L-glyS displayed nuclease resistance and maintained glycine-dependent fluorescence in serum, where the original D-glyS lost function. These results establish cross-chiral ligation as a strategy for constructing long, functional L-RNAs and broaden the possible applications of RNA-based sensors to extracellular detection of small molecules.

Detection of molecules with cell-based sensors allows for conversion of binding events into gene expression outputs. Here, we present a cell-based sensor that can detect extracellular double-stranded DNA. This sensor is based on an engineered receptor which we call Luminescent Ultrasensitive Nucleic Acid Reporter, or LUNAR. LUNAR is based on a recently developed Programmable Antigen-gated G-protein-coupled Engineered Receptor (PAGER). PAGERs are a genetic fusion of an auto-inhibitory peptide, a protein-binding domain, and a modified kappa opioid receptor. PAGERs are gated by two binding events. First, a protein ligand displaces an intramolecular inhibitor, Arodyn, then a second ligand activates the receptor. By replacing the protein-binding domain with a DNA binding zinc finger protein (ZFP) we could detect extracellular DNA in a dose-dependent fashion. Here, we show that first-generation LUNAR constructs can detect both oligonucleotides and plasmid double-stranded DNA with nanomolar sensitivity in mammalian cells. Future work will focus on improving sensitivity, fold-change, and multiplexing capabilities for sequence-specific DNA detection.

Three-dimensional protein crystals are ordered, porous macroscopic materials with potential applications in catalysis, biosensing, and biomedicine. However, most protein crystals are obtained by empirical screening, providing limited control over the lattice architecture, pore geometry or component composition that determine material function. Here, we present a modular strategy for the programmable design of highly porous, framework-like protein crystals using predefined protein-protein interactions. This strategy yielded over 30 distinct protein crystals, including single-component and multicomponent P213 and I213 lattices that grow to over 100 micrometers in size. Small-angle X-ray scattering and electron microscopy showed close agreement between experimental lattices and computational models. RFdiffusion-guided design generated isomorphous variants with matched lattice parameters, enabling coherent protein crystal alloys, epitaxial core-shell growth and reversible shell assembly. The designed crystals exhibit tunable mesoporous architectures, with limiting apertures of 2-18 nm, and support genetically encoded incorporation of fluorescent protein guests. These results establish a general route to programmable lattice engineering of protein crystals and position them as genetically encoded, compositionally tunable mesoporous materials.

Achieving predictable, tunable, and temporal control over mRNA function would grant direct regulation of gene expression, facilitating the development of new therapeutics and biotechnologies. Although several approaches for stimuli-responsive control of nucleic acids have been explored, most are limited to short oligonucleotides, lack a timed-release mechanism, or both. We envisioned a complementary method using glyoxal as a caging reagent. Glyoxal readily reacts with amidine groups found on the faces of nucleobases to give stable bis-hemiaminal adducts, directly disrupting hydrogen bonding. Fortuitously, this reaction is readily reversible, enabling spontaneous time-release decaging that varies with temperature. However, when applied previously to full-length mRNAs, the sequence length and excessive adduct formation resulted in no reactivation under relevant physiological conditions. To address this challenge, we developed chemical lithography in which portions of longer RNAs are "masked" through hybridization to complementary DNAs, permitting selective caging on only non-masked regions and preventing excessive adduct formation. We present an optimized glyoxalation protocol applied to EGFP as a model mRNA sequence and evaluate masking effectiveness through qualitative and quantitative studies. Using EGFP fluorescence, we monitored and assessed the ability of selective glyoxalation to control gene expression over time in vitro. We demonstrate the direct dependence of both the initial inhibited expression and the respective activity recovery based on the amount and location of glyoxalation. We also highlight distinct caging patterns exhibiting total inhibition upon initial treatment and complete reactivation following decaging. We anticipate that this approach will improve the mechanistic study of mRNA and gene expression and also facilitate new investigations and methods within chemical biology and biomedicine.

Membrane protein stability is strongly influenced by the surrounding lipid environment, yet how individual lipid species shape membrane proteome stability remains poorly understood. Here, we systematically examined the impact of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesteryl hemisuccinate (CHS) on membrane proteomes using membrane mimetic platforms combined with membrane mimetic thermal proteome profiling (MM-TPP). CHS shifted the proteome composition away from soluble proteins and toward integral membrane proteins, and induced concentration-dependent thermal stabilization of the mouse liver membrane proteome. Organellar membrane proteins, which displayed greater intrinsic lability than plasma membrane proteins, showed preferential stabilization by CHS. CHS supplementation of E. coli membranes similarly produced broad stabilization, indicating that this effect occurs even in cholesterol-naive systems. CHS responses were reproducible across Peptidisc and DDM and independent of CRAC/CARC motif density, supporting a broad, sequence-independent mechanism rather than selective lipid binding, although stabilization was greater among proteins with more transmembrane helices. Accordingly, individual purified proteins reconstituted with CHS exhibited only modest stabilization, consistent with a broad effect that is more apparent at the proteome scale than for any single protein examined in isolation. Together, these findings redefine CHS as a general sterol scaffold that broadly stabilizes membrane proteins and establish MM-TPP as a versatile platform for investigating lipid-dependent effects on membrane proteome stability. Subject areaIntegral Membrane Proteins, Thermal Proteome Profiling, Membrane Mimetics, Cholesterol, Lipid-Protein Interactions, Mass Spectrometry HighlightsO_LICHS broadly stabilizes membrane proteins across diverse membrane mimetics. C_LIO_LIOrganellar membrane proteins exhibit the strongest CHS-mediated stabilization. C_LIO_LICHS stabilization is conserved in cholesterol-naive E. coli. C_LIO_LIMM-TPP enables proteome-wide analysis of lipid-dependent protein stability. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/745344v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a3e017org.highwire.dtl.DTLVardef@1cd4464org.highwire.dtl.DTLVardef@1453da7org.highwire.dtl.DTLVardef@d2dc1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Molecular specificity remains a major challenge in mass spectrometry imaging (MSI), particularly when low-abundance species coexist with structurally related isomers that cannot be distinguished by accurate mass and exhibit similar fragmentation behavior. Bis(monoacylglycero)phosphates (BMPs), lysosomal lipids increasingly implicated in lipid homeostasis and disease, represent a particularly demanding example because they are structural isomers of phosphatidylglycerols (PGs) and display highly similar negative-ion fragmentation. Here, we developed an ion mobility-guided targeted MALDI-MS/MS imaging workflow for direct on-tissue discrimination of endogenous BMP/PG isomeric pairs. Orthogonal HILIC-DDA-PASEF analysis provided accurate-mass, retention-time, fragmentation, and ion-mobility information used to define mobility-constrained precursor coordinates for scheduled MALDI-iPRM-PASEF acquisition. Ion-mobility measurements showed high agreement across ESI-TIMS, MALDI-TIMS, and tissue-based MALDI-TIMS-MSI, while optimization of laser sampling minimized ion-load-dependent mobility shifts. Narrow mobility windows reduced reciprocal PG/BMP cross-talk to below 4% while preserving selective detection under strongly unbalanced abundance conditions. The workflow enabled distinct precursor- and product-ion imaging of endogenous PG 34:1 and BMP 34:1 in sagittal mouse brain, supporting their acyl-chain-level assignment as PG 16:0_18:1 and BMP 16:0_18:1. Application to a CLN3-knockout mouse model revealed BMP-specific reductions across brain, kidney, and lung that were not mirrored by the corresponding PG isomers, providing an orthogonal biological validation of the analytical discrimination. Mobility-constrained targeted MS/MS additionally resolved type-II isotopic interference that remained ambiguous at the MS1 level. Overall, this work provides a strategy for reciprocal spatial discrimination and structural confirmation of endogenous BMP and PG isomers directly in tissue and highlights the value of combining ion mobility with targeted product-ion imaging to increase molecular specificity in spatial lipidomics.