The escalating global challenge of genotoxic compounds (GCs) in environmental, pharmaceutical, and food contexts necessitates analytical approaches that combine high efficiency with independence from prior structural information. Here, we present GenoToxMass, a pioneering mass spectrometry-based predictive model that eliminates dependence on prior chemical structure. Constructed from a rigorously curated data set of 16,806 mass spectra and utilizing multimodal features, GenoToxMass achieves an area under the curve (AUC) of 0.95 with robust generalizability in external validation (AUC = 0.89). Real-world application demonstrates >85% concordance with Ames test outcomes and literature, correctly classifying 42 chemical products and 16 drug impurities in complex matrices. An openly accessible web platform enables practical deployment, while a Standard Operating Procedure (SOP) is provided for regulatory agencies. Interpretable spectral features and structural alerts enhance transparency beyond conventional "black-box" predictions, while model performance can be context-adapted through tunable decision thresholds. GenoToxMass establishes a versatile, scalable paradigm for the nontargeted screening of GCs across environmental, industrial, and regulatory landscapes, offering a new pathway that diverges from traditional animal-dependent toxicological methods.

A novel turn-on fluorescent probe 2-chloro-3-((9-(diethylamino)-5-oxo-5H-benzo[a] phenoxazin-2-yl)oxy)naphthalene-1,4-dione (NR-H2S) that could selectively response to H2S, had been designed and synthesized. NR-H2S was consist of Nile red as fluorophore and 2-chloronaphthalene-1,4-dione as receptor. Owing to the photo-induced electron transfer (PET) effect, the fluorescence of NR-H2S was quenched, while NR-H2S exhibited a remarkable “turn-on” fluorescence with the addition of H2S. NR-H2S displayed good repeatability, strong anti-ion interference ability, high sensitivity and selectivity toward H2S, which has been successfully applied to the test strips and detect H2S efficiently in food samples, showing its promising prospects.

RNA modifications are vital for all living organisms. A total of at least 63 types of uridine modification have been discovered, accounting for the largest proportion of all reported RNA modifications. Uridine modifications have also been shown to be dysregulated in various human diseases. However, it's well known that mass spectrometry analysis of uridine modifications is limited by their low ionization efficiencies. Here, we developed a simple and robust method for the simultaneous identification and quantification of 22 uridine modifications in biological samples. In fact, uridine and lots of its modifications are more easily ionized in negative ion mode, compared with positive ion mode commonly used. Besides, the fragmentation patterns of uridine modifications in negative ion mode could provide more structural information, compared with well-known glycosidic bond cleavage in positive ion mode. Furthermore, simply by adding 0.2 mM acetic acid into the mobile phase, the peak areas of uridine and all 22 uridine modifications in negative ion mode could be improved by 4.1-55.4 folds. With this novel method, we quantified 16, 14, 10, 12 and 16 uridine modifications in 293T total RNA, 293T small RNA, E. coli tRNA, S. cerevisiae tRNA and wheat germ tRNA, accordingly. This study provides a general good method for researchers interested in the identification and quantification of uridine modifications in various biological samples.

Microplastics (MPs), prevalent in water bodies, soil, and the atmosphere, pose significant risks to environmental and ecological health. By adsorbing hazardous compounds, such as organic pollutants, MPs can alter the transport and fate of these pollutants. To address this, we developed a multimodal Siamese neural network named microplastic-pollutant adsorption prediction (MPAP), trained on 1101 adsorption records covering 403 compounds and six MP types. Unlike previous models that rely on single-feature representations, MPAP leverages a multimodal architecture that integrates molecular fingerprints and graph embeddings to capture chemical structures, along with microplastic morphological features such as the MP type and particle size, as well as water chemistry parameters, enabling a more comprehensive characterization of sorption behavior. The model outperforms baseline models with R2 = 0.869 on the validation set and 0.863 on the test set. Experimental validation using batch adsorption experiments with six previously untested pollutants, quantified via liquid chromatography-mass spectrometry or microwave plasma torch ionization-mass spectrometry in different environments, confirmed a strong predictive performance. To support broad application, we provide an open-access web platform (http://mpap.envwind.site:8004/) for rapid, high-throughput prediction across diverse MP-pollutant-water environment scenarios.

An in-depth study of electrochemistry (EC) requires a sensitive and real-time monitoring platform. Herein, we report a bubble extraction ionization electrochemical mass spectrometry (BEI-EC-MS) platform for monitoring the reaction products and transient intermediates in the EC reactions. Compared with previous online EC-MS methods, BEI-EC-MS employs in situ soft ionization within the electrolytic cell, eliminating the need for additional voltages during the reaction monitoring process. The ascending bubbles effectively extract and concentrate compounds from both the surface of the EC electrode and electrolyte, enabling long-term monitoring in both positive and negative MS modes. Monitoring experiments of aniline (ANI) EC polymerization verified the platform performance and successfully captured short-lived radical intermediates. The platform was then used to monitor the long-term EC degradation of the pollutants dinotefuran (DNT), rhodamine B (RhB), and perfluorooctanoic acid (PFOA). A series of intermediates and products were characterized by using BEI-EC-MS, including various radical ions measured for the first time, providing pivotal evidence for the degradation mechanisms of these reactions. We anticipate that our approach will not only provide new protocols for monitoring EC reactions and assist in conducting mechanistic studies but also be readily adaptable in conventional electrochemical laboratories with the mere addition of a bubble generation device.

The proliferation of phosphodiesterase-5 inhibitor (PDE5I) derivatives in food products and environmental samples poses a growing health concern, as these illicit additives (IAs) are often designed to evade conventional targeted detection methods. A cheminformatics tool capable of rapid and accurate screening of PDE5Is is therefore urgently needed for both food safety and environmental monitoring. To address this need, we developed TransIA (Transformer-based Illicit Additives Detection Network), which identifies PDE5Is from raw mass spectra. The model achieved 98% accuracy on the testing set with a false-positive rate below 3% across diverse environmental and biological matrices. Deployment across 197 real-world samples facilitated the identification of 21 IAs, of which 16 were previously unseen by our model (5 from the test set, 7 catalogued in PubChem but absent from our database, and 4 entirely unreported). This approach may offer a theoretical framework for integrating the model with portable mass spectrometry to assist on-site environmental analysis.

Metal ions are indispensable cofactors governing protein conformations and biofunctions. However, given the highly dynamic and subtle nature of metal ion modulations, capturing the metal-induced conformational dynamics and resolving these structural alteration details remain challenging using traditional structural biology methods. Herein, we apply native mass spectrometry (nMS) and 193-nm ultraviolet photodissociation (UVPD) to characterize the metal ion-dependent conformations of Kirsten rat sarcoma (KRAS). Achieving high sequence coverage, we established a biophysical baseline for the native Mg2+ cofactor, demonstrating how its structural impact modulates functional switch loops to stabilize a compact and inactive state of KRAS. Furthermore, we find the competitive substitution of Mg2+ by Zn2+ preserves the global compact topology but alters local stability, imparting the active site and switch regions with improved flexibility. Our work highlights how metal ion modulates on KRAS conformational dynamics, providing a sensitive MS-based analytical strategy for exploring the allosteric modulations across broader metal-proteins.

Understanding the functions and regulatory mechanisms of the epitranscriptome entails robust and accurate analytical methods to identify and quantify post-transcriptional modifications in RNA. However, there are still various challenges in analyzing multiple modified nucleosides in RNA. Herein, we established a highly sensitive and high-throughput hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method, in conjunction with a stable isotope-dilution technique, for accurate quantification of 35 nucleosides. By the use of malic acid as a mobile phase additive, the HILIC-based separation of nucleosides was improved and the MS signal response of nucleosides was enhanced by 2.5- to 20.0-fold. Notably, seven groups of isomeric nucleosides with identical multiple-reaction monitoring ion transitions and six groups of nucleosides with identical or similar molecular weights that were indistinguishable by MS were well resolved by optimal HILIC separation. Thirty-five nucleosides were analyzed simultaneously within 12.5 min, and the limits of detection of these nucleosides ranged from 15.0 amol to 43.5 fmol. With this method, we conducted a comprehensive analysis and evaluation of the alteration in the RNA modification profile in breast cancer and assessed the RNA modification patterns across different breast cancer subtypes. The developed HILIC-MS/MS method has excellent capabilities for sensitive and high-throughput detection of multiple modified nucleosides, thereby providing a valuable analytical tool for deciphering the epitranscriptomic landscape and screening nucleosides as biomarkers in future clinical research.

RATIONALE:The comprehensive quality control of chiral pharmaceuticals like mitiglinide necessitates simultaneous assessment of chemical impurities and enantiomeric purity, yet conventional workflows address these separately, leading to inefficiency. This study develops a comprehensive analytical strategy to overcome this challenge for the anti-diabetic drug mitiglinide. METHODS:For chiral analysis, an online heart-cutting two-dimensional liquid chromatography-high-resolution mass spectrometry (2D-LC-HRMS) method was developed. First, impurity profiling of mitiglinide was accomplished using a one-dimensional reversed-phase LC-HRMS (1D-LC-HRMS) method. Subsequently, the 2D-LC-HRMS system achieved enantiomer separation by online coupling of a C18 column (first dimension) with a polysaccharide-based chiral column (second dimension), with the separated analytes detected by an Orbitrap mass spectrometer. RESULTS:1D-LC-HRMS identified five major impurities, structurally characterizing four, with the main component accounting for only 49.12% of the total integrated peak area (relative abundance by EIC peak area normalization, not absolute purity). The 2D-LC-HRMS method achieved effective enantiomer separation. A consistent third minor chromatographic peak was observed across six replicate analyses, which is tentatively assigned as a potential diastereomeric impurity based on stereochemical interpretation of the chromatographic behavior; confirmatory evidence is required for definitive identification. CONCLUSIONS:This work successfully establishes a comprehensive strategy that efficiently consolidates impurity profiling and chiral purity assessment for mitiglinide. It provides a reliable, more informative approach for the quality control of complex chiral pharmaceuticals.

Water microdroplets have emerged as extraordinary reaction vessels in which ultrahigh electric fields at the air-water interface generate reactive radicals and hydrated electrons, enabling transformations inaccessible in bulk solution. However, microdroplet-induced free-radical polymerization has remained virtually unexplored. Here we demonstrate that aqueous microdroplets induce the rapid, room-temperature polymerization of carbon disulfide (CS2) into a poly(carbon sulfide) solid. The reaction consumes CS2 completely without catalysts at room temperature. Comprehensive characterization reveals a highly cross-linked polymer composed of conjugated C═C and C-S networks with incorporated hydrogen. Radical quenching and online mass spectrometry experiments reveal a mechanism involving electron transfer at the droplet interface, initiating stepwise CS2 radical polymerization pathway. Gram-scale preparation is readily achieved through through small-scale parallel reactions. This discovery establishes first microdroplet-mediated polymerization for polymer synthesis and offers a viable strategy for CS2 pollutant valorization.

Weak and transient molecular interactions can play crucial roles in various biological and chemical processes but are not easy to study with classical structural methods. Herein, we explore the weak interactions between an artificial triplet photoenzyme, RamR, and two triplet quenchers using native mass spectrometry (nMS). The quenchers significantly affect the enzyme reaction, yet the molecular mechanism remained elusive. We systematically analyzed the protein-quencher complexes in both positive and negative ion modes, revealing small charge state shifts indicative of molecular interactions. Collision-induced dissociation (CID) and surface-induced dissociation (SID) were employed to investigate the binding strength, revealing weak binding without well-defined stoichiometry. Interestingly, the addition of both quenchers resulted in shaper peaks in nMS, suggesting competitive binding with residual buffer contents in solution. Combined with previous functional assays, our result agrees with the hypothesis that the quenchers interact with hydrophobic regions of RamR, potentially altering the protein's surface charge and activity. The findings showed that subtle spectral features in nMS can offer clues for investigating protein-ligand binding involving weak or transient interactions.

Chirality is essential for life, as molecular asymmetry underpins DNA structure, enzyme specificity, and biochemical reactions, highlighting the critical need for precise chiral analysis in fields such as medicine and drug development. In this perspective, we have summarized the development of chiral mass spectrometry instrumentation over the past two decades and present our perspectives and outlook on future directions. This manuscript focuses on reviewing five approaches for achieving mass-selective chiral analysis, including tandem mass spectrometry, ion mobility spectrometry, mass-select photoelectron circular dichroism, mass-resolved photoelectron circular dichroism, and directional rotation mass spectrometry. Methods lacking instrumental innovation, such as traditional chiral chromatography, are not discussed herein.

Hypertension remains a global health burden, driving demand for effective therapies. Short bioactive peptides offer high specificity and low toxicity for antihypertensive treatment, but their discovery traditionally relies on low-throughput extraction from natural proteins. Here we introduce PepGate, a gated dual-path discrete diffusion framework for de novo ACE inhibitory peptide design. By fine-tuning a protein language model into a short-peptide-specific foundation model (PepGPT), we developed an integrated pipeline comprising a generative diffusion model (PepGen) and discriminators (PepClass and PepIC50). PepGate identified 30 promising peptides with a median IC50 of 4.79 μM. In vivo validation of the top candidate, YIPVPF, demonstrated sustained blood pressure reduction in SHRs (acute SBP reduction: 48 mmHg; chronic: 43 mmHg). Computational target-network analyses further suggested broader blood-pressure-related mechanisms beyond ACE inhibition. This pipeline shifts from empirical mining to precision digital design, offering a scalable tool for next-generation therapeutic peptide development.

Lung cancer remains the leading cause of cancer-related mortality. The rising incidence among never-smokers underscores the role of environmental exposures, particularly contaminants of emerging concern (CECs) -a diverse group of largely unregulated chemicals with potential carcinogenicity. Yet, their links to lung cancer risk and prognosis are not well defined. To address this, we developed a robust and sensitive pseudo-targeted LC-MS/MS exposomics platform using 97 reference standards to semi-quantitatively profile 350 serum CECs in a hospital-based cohort comprising 570 lung cancer patients and 307 healthy controls. The method demonstrated high analytical reliability detecting 228 CECs across all participants. Several compounds-including monomethyl phthalate (MMPA), perfluorooctanesulfonic acid, and simazine-were significantly elevated in patients. Mixture models confirmed synergistic effects of co-exposures, and among 75 postoperative recurrence cases, MMPA, bisphenol G, and Irganox 245 emerged as. Key recurrence-associated chemicals. Absolute quantification revealed significantly higher serum MMPA levels in patients (83.09 μg/L) compared to controls (57.19 μg/L). Proteomic profiling of MMPA-exposed A549 lung cancer cells showed dysregulation in pathway related to chromatin remodeling, autophagy, cytochrome P450 metabolism, and immune function. This study integrates exposomics and proteomics to identify CECs linked to lung cancer development and recurrence, offering novel insights into environmental contributions and potential molecular targets in disease progression.

A phosphine-catalyzed three-component cyclization reaction between anilines, carbon dioxide, and chloroalkanes was developed for the synthesis of oxazolidinones. This strategy not only proceeds under ambient CO2 pressure and metal-free condition but also shows a broad substrate scope, including aromatic amines, aliphatic amines, chiral amino acid esters, and bioactive molecules, providing an efficient and environmentally benign route to synthesize pharmaceutically relevant N-aryl-oxazolidinones. Mechanistic investigations utilizing mass spectrometry (MS) indicate the involvement of multiple phosphine intermediates in this process, thereby elucidating the underlying mechanism. Moreover, the relationships between these phosphine intermediates and Tolman cone angles or the solvent effect of phosphines were examined through mass spectrometry.

Quinones are highly reactive oxidants that pose risks of cytotoxicity and genotoxicity to the human body. Sensitive analysis of quinone pollutants by mass spectrometry remains challenging due to the very limited ionization efficiency of quinones and the long pretreatment time. Here, we developed a rapid and highly sensitive in-source microdroplet derivatization strategy for the determination of quinones in complex matrices. Triphenylphosphine was used as a novel "tag" to react with quinone for online derivatization of quinones via the microdroplet-driving conjugate addition reaction. The formation of phosphonium ions significantly increased the ionization efficiency of the quinones. The sensitivity was improved by 2-3 orders of magnitude as compared with other online derivatization methods. Optimization results showed that the nebulization gas pressure, capillary temperature, sample solvent concentration, and reagent concentration played important roles in the derivatization and ionization of the quinones. The developed method featured good linearity (R2 ≥ 0.996), high sensitivity (LODs at the nmol/L level), and qualified precision (RSDs ≤ 7.3%) for four typical quinones. The method was successfully applied to the determination of quinones in complex matrices including human serum and urine. Taking into consideration the fact that many derivatization procedures are completed in bulk solutions, the proposed in-source microdroplet derivatization method offers us an effective way for fast screening of derivatization reagents. The present work expands the utility of microdroplet chemistry as well as chemical derivatization in the analysis of trace compounds, which might have potential applications in the fields of environmental sciences and clinic analysis.

ABSTRACT Cembranoids are significant flavor precursors found in flue‐cured tobacco leaves. The degradation products can serve as important aromatic compounds. In this study, 16 cembranoids were discovered in flue‐cured tobacco leaves by liquid chromatography–mass spectrometry (MS), and 1 cembranoid featuring both oxidation and dehydrogenation from α / β‐cembranoid was identified for the first time. α‐ and β‐cembranoids were confirmed to be precursors to various degradation products. Additionally, β‐cembranoid was prone to epimerize into α‐cembranoid while simultaneously undergoing allylic rearrangement, with the hydroxyl group undergoing migration. During the aging and curing processes, cembranoids can easily add oxygen atoms to the carbon–carbon double bonds, whereas the hydroxyl group in the structure may form epoxides, oxidize into ketones, or undergo dehydration reactions. Additionally, the microwave plasma torch interfaced with an MS was used to create and detect the pyrolysis and oxidized products of cembranoids. More degradation products were discovered, including oxidation products with the addition of one to three oxygen atoms, as well as a series of aromatic compounds such as 1,3,5‐heptatriene and its range of homologs. This research presented the different degradation pathways of cembranoids in flue‐cured tobacco during aging and curing processes, offering valuable insights for the enhancement of tobacco production techniques and quality control.

Background: Oligonucleotide-based drugs have gained significant attention as a promising class of therapeutics due to their precise ability to regulate gene expression. However, the presence of impurities in these drugs can compromise their safety, efficacy, and stability. Therefore, the identification and detailed analysis of these impurities are crucial. Traditional methods may struggle to detect subtle impurities, such as oxidation and hydrolysis products, which necessitate the development of more advanced and reliable analytical techniques for oligonucleotide therapeutics. Results: In this study, we introduce a novel analytical method that combines heart-cutting two-dimensional liquid chromatography (2D-LC) with tandem mass spectrometry (MS/MS) to effectively separate, analyze, and identify impurities in oligonucleotide-based drugs. The first dimension utilizes anion exchange chromatography (AEX) to separate oligonucleotides based on their negative charge, while the second dimension employs reverse ion-pair chromatography (RIPC) for further purification and compatibility with mass spectrometry. This 2D-LC-MS/MS approach provides a sensitive and accurate means of identifying and quantifying impurities, including oxidation and hydrolysis products. The method was applied to two RNA interference (RNAi) drugs, Givosiran and Patisiran, where 3 and 20 impurities were identified, respectively. Additionally, sequencing analysis using data dependent acquisition (DDA-MS/MS) enabled the determination of the molecular weight and structural characteristics of these impurities, offering a comprehensive and detailed view of the impurity profiles. Significance: This novel heart-cutting 2D-LC-MS/MS method offers a significant advancement in the analysis of impurities in oligonucleotide-based therapeutics. It provides high sensitivity, enabling the identification of subtle impurities that are often challenging to detect. By applying this technique to RNAi drugs, we demonstrate its potential to enhance the safety, efficacy, and stability of oligonucleotide-based therapies, making it a valuable tool for the pharmaceutical industry.

The metabolism process of amino acids is closely related to the growth of normal and cancer cells. It is still not clear how L/D-configuration amino acids participate in the metabolism of colorectal cell. Herein, intra- and extra-cellular metabolic distribution of L/D-amino acids in colorectal cell (HCT116) and human normal intestinal epithelial cell (NCM460) were profiled utilizing HPLC-MS/MS coupled with a chiral probe. The results displayed the differential metabolic portrayal for the two cell lines. Compared with NCM460 cell, 13 kinds of significant differential amino acids were founded in a lower concentration within HCT116 cell, and L-Gln was even not detected for intra-cell; as for extra-cell culture medium, the HCT116 cell consumed more L-Gln, D-Phe and D-Leu, while L-Met was low ingested in HCT116 cell. L-Ala and Gly were excretion in both two cell lines, excepted L-Cit which was uptake in HCT116 and excretion in NCM460 cell. Furthermore, the dynamic changes of chiral amino acids displayed that phenylalanine, tyrosine and tryptophan biosynthesis and arginine biosynthesis is the major pathway for intra-cellular metabolites and extra-cellular metabolites, respectively. Moreover, with additional D-amino acids in culture medium, the results exhibited that high concentration of D-amino acids have no significant effect on the proliferation of NCM460 cell, but could influence the profiling of amino acids metabolites, and further affect the proliferation of HCT116 cell. This present work enhances the understanding of these differential amino acids metabolic network and depicts a dynamic process of metabolic dysregulation of HCT116 and NCM460 cell.