JACS Au

Platinum anticancer drugs tend to target DNA whereas certain ruthenium and osmium organometallic compounds, including those with known anticancer activity, preferentially bind histone proteins in chromatin. We earlier found that Ru/Os arene 2-pyridinecarbothioamide antitumor agents display unique or partially overlapping profiles of histone protein binding in the nucleosome compared to Ru arene phosphaadamantane (RAPTA) antimetastasis drugs, but the basis for this difference is unclear. Here we structurally characterized the nucleosome binding effects of arene ligand substitutions and carried out a multiscale simulation analysis, which reveals that the interplay between metal cation and non-leaving ligand identity dictates adduct stability and whether complexes target electronegative surface patches, internal crevices, or both. We show that the nucleosome superhelical crevice acts as a small molecule selectivity filter and that multi-site binding profiles can be expanded or reduced through defined ligand substitutions, which modulate dynamic and steric attributes. Our findings suggest new avenues for rationally developing Ru/Os organometallics that could help expand the scope of chromatin-targeting therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/688318v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@188bc1dorg.highwire.dtl.DTLVardef@1f619d5org.highwire.dtl.DTLVardef@1a0ebaorg.highwire.dtl.DTLVardef@bd0abb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Affinity purification is a essential technique for isolating highly purified proteins; however, generating affinity ligands require significant time and financial investment. To address these limitations, this study proposes a novel affinity chromatography method utilizing in silico-designed cyclic peptides as ligands. Targeting Complement C1q (C1q), a plasma protein that plays crucial roles in classical complement pathway, we employed the biomolecular structure prediction model, AlphaFold2, to design specific binding cyclic peptides. Based on these designs, we synthesized lariat-type cyclic peptides characterized by disulfide cyclization and biotinylation, which were subsequently immobilized on streptavidin carriers. Performance tests confirmed that the resulting column specifically captured C1q, allowing for elution via a standard NaCl concentration gradient. Notably, high selectivity was preserved even in the presence of plasma, underscoring the ligands practical robustness. By overcoming traditional constraints through (1) rapid and simple design, (2) high specificity, and (3) universal versatility without genetic modification, this de novo design strategy represents a potential breakthrough in protein purification technologies. HighlightsO_LIAI-driven de novo design generated a specific cyclic peptide ligand for Complement C1q C_LIO_LIThe synthetic ligand enabled one-step purification of Complement C1q directly from human plasma C_LIO_LIMild elution conditions preserved the targets oligomeric structure and native interactome C_LIO_LIThis label-free strategy offers a rapid, low-cost alternative to antibody-based chromatography C_LI

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of six novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Among the candidate enzymes, u17, u10, and u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Building on these results, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. Collectively, these findings highlight amidase signature urethanases as valuable scaffolds for advancing sustainable and scalable biocatalytic recycling of polyurethanes. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/705263v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@127bf23org.highwire.dtl.DTLVardef@75c29corg.highwire.dtl.DTLVardef@13bbf30org.highwire.dtl.DTLVardef@18504a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

GPR52 is an orphan G protein-coupled receptor implicated in psychiatric and neurodegenerative disorders, but its endogenous ligand remains unidentified, limiting the exploration of its physiological functions and therapeutic potential. We pioneered a novel methodology for orphan GPCR ligand discovery utilizing the GPCR-activation-based (GRAB) strategy by developing GPR52-1.0, a genetically encoded fluorescent sensor. GPR52-1.0 exhibits excellent membrane trafficking and high sensitivity in HEK293T cells, cultured neurons, and acute mouse brain slices. Notably, it detects neuronal activity-dependent endogenous ligand release in the striatum, with responses abolished by a specific antagonist. This sensor provides a powerful tool for identifying GPR52s endogenous ligand(s) and enables real-time monitoring of its activation. Our work lays the foundation for uncovering GPR52s physiological roles and supports future efforts to develop GPR52-targeted therapeutics.

The protein tyrosine phosphatases (PTPs) SHP-1 and SHP-2 play complex roles in a variety of signaling pathways, including those involved in cancers and other diseases, making them important drug targets. These two PTPs have superimposable active sites, but different biological functions in vivo, including opposing roles in cancer development. Unique to these PTPs is the presence of two tandem Src homology 2 (SH2) domains, which regulate access to the phosphate binding site in the catalytic domain, through an autoinhibition mechanism. Studies of the allosteric regulation and dynamics of these PTPs, as well as associated drug discovery efforts, typically focus on autoinhibition rather than the dynamics of a catalytic loop in the phosphatase domain, the WPD-loop, which is essential for PTPase activity. However, recent deep mutational scanning data has demonstrated that oncogenic mutations also regulate WPD-loop motion in SHP-2. We provide here a detailed computational study of WPD-loop dynamics and catalysis in wild-type and mutant full-length and truncated (catalytic domain only) SHP-1 and SHP-2, demonstrating that many oncogenic residues lie on the allosteric pathways regulating WPD-loop dynamics. Mutations at these positions alter WPD-loop dynamics, disrupting the active site and negatively impacting catalysis. Further, our simulations provide molecular insight into the link between the presence of the SH2 domains and loop motion in the catalytic domain, and, importantly, how it differs between the two PTPs. Taken together, our work showcases the impact of altered WPD-loop motion in oncogenic SHP-1 and SHP-2 variants, opening new strategies for selectively targeting these important therapeutic enzymes.

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sucrose synthase (SuSy) has been suggested as a key enabling enzyme for uridine diphosphate glucose (UDP-Glc) regeneration in glycosyltransferase-catalyzed biotransformations. However, its stability and efficiency in industrially relevant conditions have not been characterized or engineered, limiting its industrial readiness. Here, we combined enzyme discovery and characterization with comprehensive semi-rational enzyme engineering strategies, to optimize SuSys catalytic activity, thermostability, solvent tolerance, and soluble expression. The engineered variants were significantly more stable than wild-type, with up to 13.6 {degrees}C increase in melting temperature, over two orders of magnitude improvement in half-lives at elevated temperatures, and approximately three orders of magnitude increase in total turnover number. Additionally, the optimized variants retained up to 75% relative activity at 60 {degrees}C in the presence of 25% (v/v) DMSO, which the wild-type shows near complete loss of activity. Structural and molecular dynamics analyses reveal how mutations modulate conformational dynamics and hydrophobic packing, favoring catalytically competent conformations. Using methyl anthranilate glycosylation as a representative biotransformation, we demonstrate that the engineered SuSy variants consistently outperform both wild-type SuSy and stoichiometric UDP-Glc systems, enabling efficient UDP-Glc regeneration at reduced enzyme and sugar donor loadings. Finally, techno-economic and environmental assessments further indicate that implementation of engineered SuSy reduces reaction cost by approximately 6- and 2-fold relative to UDP-Glc and wild-type systems, respectively, while achieving average reductions of 3- and 2-fold in environmental end-point impacts. These results established SuSy engineering as a critical enabler for sustainable glycosylation reactions.

Histamine is a key signaling molecule in pathophysiology that can exhibit significant regulatory roles in diverse health and disease status. Besides the well-studied noncovalent interactions between histamine and its receptors, protein histaminylation is a recently discovered mode of action, through which histamine regulates cellular signaling pathways in a covalent-interaction manner. Histaminylation is an emerging protein post-translational modification, where an isopeptide bond is formed between the histamine primary amine and {gamma}-carboxyl group of glutamine through a transamidation reaction catalyzed by transglutaminase 2 (TGM2). However, due to the lack of efficient pan-specific antibodies targeting histaminylated glutamine, the histaminylation proteome in cells remains poorly explored. Here, we report the design and development of a novel N{tau}-propargylated histamine probe as well as its successful application in chemical proteomic profiling of the histaminylation proteome in cancer cells. Notably, new TGM2-catalyzed epigenetic marks on core histones, e.g., H2AXQ84 and Q104 histaminylation, have been identified from cancer cells and verified in this study.

Douglass Cooperativity and Ciullis Cooperativity in induced-proximity systems, remains controversial with paradoxes such as path-dependent metrics and apparent universal negative Cooperativity. We noticed that in "partial-embedded" model, a substantial portion of giant ligand remains exposed outside and does not engage with the host proteins force field. It incurs an entropic cost due to the restriction of translational/rotational degrees of freedom. This large, mass-dependent unfavorable ligand entropy penalty normally shifts binding affinity to 104[~]108-fold. ITC thermodynamic cycles analysis confirmed the dramatic entropy loss among reaction pair. This reconciles the conflicting Cooperativity definitions, yielding true path-independent positive PPI Cooperativity from observed entropy loss subtracting ligand entropy penalty. ITC data showed rigid linkers appear superior to flexible linkers with respect to both oral bioavailability and safety profile in PROTAC design. "ligand entropy barrier wall/Cooperativity ladder" pair is not only impact induced-proximity systems but also constitute the physical basis for all biosystems.

Untargeted proteomics enables quantitative determination of host cell proteins (HCPs) in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. We report a prospective validation of label-free untargeted proteomics for HCP quantification using a total-error (TE) approach. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20-80 ng). Four independent assays (198 injections) supported hierarchical replication and one-way random-effects ANOVA variance decomposition with Welch-Satterthwaite adjustment. Dual entrapment analysis demonstrated empirical peptide-level false discovery proportions below 1% at q = 0.01. Deterministic parsimony inference ensured invariant protein-group definition. Weighted least-squares regression (R{superscript 2} = 0.993) identified stable proportional compression with recoveries of 81-85%. Repeatability dominated the variance structure (median CV 2.7%); intermediate precision total SD ranged from 0.69% to 3.81% over the validated range. Accuracy profiles integrating empirical bias with a log- log variance model showed 95% {beta}-expectation and 95/95 content tolerance intervals fully contained within {+/-}30%, with a lower limit of quantification (LLOQ) of 20 ng. Abundance-stratified TE analysis revealed concentration-dependent calibration heterogeneity masked by aggregate-level estimation; stratum-specific {beta}-expectation intervals within {+/-}35% defined an abundance-aware LLOQ of 3.6 ppm (P95 = 3.87 ppm). Robustness under independent search software (FragPipe, CCC = 0.998, LoA {+/-}9%) and cross-platform acquisition (Astral, CCC = 0.980, LoA {+/-}18%) remained within predefined {+/-}30% agreement limits. System suitability criteria were derived empirically from validation performance. This is the first prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification, with a statistical framework applicable to other high-dimensional analytical methods requiring regulatory qualification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/710150v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f5331aorg.highwire.dtl.DTLVardef@ee2234org.highwire.dtl.DTLVardef@798eaorg.highwire.dtl.DTLVardef@c84034_HPS_FORMAT_FIGEXP M_FIG C_FIG

The structural study of membrane proteins has traditionally relied on detergent-based extraction from cellular membranes. Although native-like reconstitution approaches have advanced, fully understanding membrane protein dynamics requires examining them within their native membrane environment. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for probing structural dynamics in reconstituted systems, but the presence of the lipid bilayer introduces considerable complexity, limiting broader adoption under physiological conditions. Here, we present the first fully automated HDX-MS platform incorporating a two-stage delipidation workflow. We applied this approach to monitor the dynamics of the ABC transporter MsbA in isolated inner membrane vesicles (IIMVs) from Escherichia coli through its ATPase cycle. IIMVs revealed distinct dynamic features within the nucleotide binding domains and substrate binding cavity, highlighting physiologically relevant motions not observed with detergent solubilised MsbA. This platform significantly advances HDX-MS and underscores the importance of studying membrane proteins in native lipid environments.

P(V) electrophiles such as tabun, sarin, soman, and VX are notorious for their lethality and nefarious intent in chemical warfare. Consequently, these deadly agents have largely been abandoned except for fluorophosphonate tool compounds that were repurposed for activity-based protein profiling (ABPP). Stereogenic P(V) centers hold strong potential as enabling scaffolds for synthetic and medicinal chemistry due to their inherent chirality and favorable bioavailability but are limited principally by potent off-target toxicity. Herein, we developed phosphorus-azole exchange (PhAzE) chemistry for tuning reactivity of the stereogenic P(V) pharmacophore to increase selectivity and mitigate off-target activity in cells and animal models. We demonstrate ultrapotent (300 pM in cells, 1 mg kg-1 in mice), enantioselective, covalent inhibition of the serine hydrolases DPP8/9 with PhAzE ligand in cells and in vivo; no overt toxicity was detected in mice treated daily over the course of a week. These finding show the P(V) electrophile can potently and enantioselectively engage a target protein without a deadly outcome, charting a path towards broader adoption of these agents in laboratory and industry settings.

Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM). L,D-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of {beta}-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.

The G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) is a key mediator of bile acid signaling, exerting its physiological effects through coupling with the stimulatory G protein (Gs). This interaction is essential for stabilizing the receptors active conformation and triggering downstream signaling. Among endogenous ligands, lithocholic acid (LCA) is the most potent natural agonist. However, the dynamic features underlying its binding and activation mechanisms remain poorly defined. In this study, we investigated the molecular basis of the interaction between LCA and GPBAR1, as well as the functional consequences of this interaction on receptor activation by integrating homology modelling, molecular docking, and molecular dynamics (MD) simulations. Our calculations reveal that LCA binding stabilizes the active state of GPBAR1, biasing the conformational ensemble of TM5 and TM6, as well as the main microswitches. These ligand-induced rearrangements enhance the coupling interface with the 5 helix of Gs and facilitate allosteric communication between the orthosteric and intracellular sites. Overall, our findings provide dynamic insight into how LCA modulates GPBAR1 activation and G protein engagement, highlighting its role as a molecular effector in bile acid signaling, and furnishing molecular detail relevant to ongoing efforts in GPBAR1-targeted compound development.

The conformational dynamics of a model cationic protein in water and in the presence of anionic sodium dodecyl sulphate (SDS) and cationic cetyltrimethylamonium bromide (CTAB) surfactants at different concentrations were investigated using all-atom molecular dynamics simulations. Free-energy landscapes constructed along principal components reveal a compact, well-defined native basin at 25 {degrees}C in water, whereas elevated temperature (100 {degrees}C) induces a broadening of the conformational space and the emergence of multiple metastable states. The presence of surfactants further modulates this behavior in a concentration-dependent manner. Cluster population analysis shows that SDS promotes a highly heterogeneous ensemble characterized by reduced dominance of the native-like cluster, while CTAB partially protects the protein from thermal denaturation at higher concentrations. Radial distribution functions demonstrate strong accumulation of SDS headgroups around the protein and pronounced insertion of SDS alkyl tails into hydrophobic protein regions, indicating direct hydrophobic destabilization and micelle-assisted unfolding. In contrast, CTAB exhibits weaker headgroup association owing to electrostatic repulsion and reduced tail-hydrophobic contacts, suggesting a less disruptive interaction mechanism. At high concentration, CTAB aggregates provide a structured hydrophobic environment that stabilizes the folded state and suppresses denaturation. Together, these results provide a molecular-level picture of how surfactant chemistry and concentration govern the conformational stability of a cationic protein, highlighting the dominant role of hydrophobic interactions in surfactant-induced denaturation at high temperature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/717321v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@f68004org.highwire.dtl.DTLVardef@14e9a98org.highwire.dtl.DTLVardef@18771d3org.highwire.dtl.DTLVardef@141fc6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Six-transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) has emerged as a promising therapeutic target for prostate cancer. We have optimized the expression and purification conditions of human STEAP1 to maximize the production of its homotrimeric form, which is crucial for metal ion reduction and maintaining cellular redox balance. Proteins obtained from these optimized conditions were complexed with both heme and flavin-adenine dinucleotide (FAD), two cofactors that are fundamental to STEAP functionality, suggesting native folding and interactions of the protein. In addition, we compared the impact of stable and transient expression systems on the protein quality of STEAP1. We found that stable expression promoted heme incorporation, improved expression homogeneity, and ensured correct protein orientation on cell surfaces. Our findings present effective strategies for optimizing the recombinant production of STEAP1, with potential applicability to other STEAP family proteins to facilitate therapeutic discovery.

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease. Mass spectrometry-based analysis is the gold-standard for histone PTM analysis, but it remains constrained by inefficient sample preparation workflows requiring multiple days. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation from days to hours while improving PTM coverage and quantitative accuracy. Through systematic evaluation of the Arg-C Ultra protease and a prototype recombinant (r)-Chymotrypsin protease under varied conditions, such as chemical derivatization using propionic anhydride and tandem mass tags (TMT), we demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM comparable to conventional Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMTs tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 50 succinylation and 27 glutarylation sites - a dark epigenome largely undetected by propionylation-based methods. We demonstrated that complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identifies >200 PTMs including biologically critical PTM sites H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A ubiquitination at K118/K119. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.

Ambient metabolomics techniques such as laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS) enable fast, preparation-free fingerprinting of biological samples but are inherently limited by spectral congestion in the absence of chromatographic separation. While ion mobility spectrometry provides additional gas-phase separation, maintaining ion transmission under the transient signals characteristic of laser desorption, remains analytically challenging. Here, we define operating conditions for cyclic traveling-wave ion mobility spectrometry (cIMS) that preserve transmission under LA-REIMS duty-cycle constraints and systematically evaluate how cIMS integration reshapes biofluid fingerprints and enhances chemical specificity in chromatography-free metabolomics analysis. Under optimized single-pass conditions, cIMS separation reorganized LA-REIMS spectra into structured mass/mobility feature domains, enabling selective mobility-based filtering of matrix-derived salt cluster ions. This reduced non-biological background contributions by up to 35% of total spectral intensity while preserving over 90% of detected untargeted features. Although cIMS operation introduced a sensitivity penalty relative to time-of-flight-only acquisition, approximately 80% of the total ion current was recovered under optimized conditions. Mobility-resolved data revealed coherent homologous series and class-specific structural trends, particularly for lipids, supporting class-level annotation. Analysis of 101 metabolite and lipid standards covering a broad physicochemical range (logP -5.30 to 19.40) demonstrated comprehensive molecular coverage, high mass accuracy (mean 2.4 ppm), and good agreement with reference CCS values (mean deviation 4.0%), with isomer separation observed for biologically important secondary bile acids in extended separation cycles. Collectively, these results establish LA-REIMS-cIMS as a practical analytical strategy for enhancing chemical specificity and spectral interpretability in support of high-throughput large-scale metabolic fingerprinting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/709786v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@18a2dfdorg.highwire.dtl.DTLVardef@d165d6org.highwire.dtl.DTLVardef@1750291org.highwire.dtl.DTLVardef@fbbce9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Ion mobility spectrometry adds an orthogonal gas-phase separation to LA-REIMS, reorganizing complex biofluid spectra into distinct mass-mobility feature bands and improving molecular resolution in rapid ambient ionization metabolomics. C_FIG

Rapid and highly selective sensing of ultra-low concentration protein biomarkers remains a critical challenge important for early disease diagnosis and monitoring. Here, we use conical SiO2 nanopore-based biosensing for the rapid detection of heart-type fatty acid binding protein (H-FABP). Antibodies were covalently immobilized on the nanopore surface through siloxane chemistry. The functionalized asymmetric nanopores generate a characteristic rectifying current-voltage response, which shows a distinct shift upon binding to the target protein due to partial neutralization of the negatively charged pore surface. The sensor exhibits excellent sensitivity in the attomolar to nanomolar concentration range with a detection limit (LOD) of [~]0.4 aM. Furthermore, the platform exhibits high selectivity, distinguishing H-FABP from non-target proteins (HSA and Hb) at concentrations six orders of magnitude higher. We also demonstrate that nanopores can be regenerated using sodium hypochloride and O2 plasma treatment, enabling repeated functionalization and reuse.

Kisspeptins are the small peptide products encoded by the KISS1 gene and physiologically exist in various isoforms of variable length. They are the central regulators of reproduction, being a prominent driver of GnRH hormone secretion. Additionally, they have emerged as an important peripheral therapeutic target for many metabolic diseases like diabetes, obesity, and polycystic ovary syndrome (PCOS). Despite their therapeutic potential, their utility is severely limited by their short half-life. We have rationally bioengineered two versions of native kisspeptins, which we named HSK-1 and HSK-2. HSK-1 (8kDa) and HSK-2 (13kDa) are derived from the fusion of the albumin-binding ZAG domain from Streptococcus zooepidemicus with KP-10 and KP-52 versions of kisspeptins (KPs), respectively. In vitro assays confirmed that the proteins were functionally active and triggered downstream signalling. Molecular dynamics simulations of the proteins revealed their structural features relative to the native kisspeptin isoforms. Both molecules demonstrated stable receptor engagement, and ligand-induced conformational changes were observed, suggesting receptor activation. HSK proteins demonstrated an extended half-life and mostly acted peripherally in young animals. They reduced peripheral luteinizing hormone levels in young animals, likely representing a previously unrecognized mode of peripheral kisspeptin action. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/703727v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@7b02fdorg.highwire.dtl.DTLVardef@13da0org.highwire.dtl.DTLVardef@174d467org.highwire.dtl.DTLVardef@124c653_HPS_FORMAT_FIGEXP M_FIG C_FIG