Pseudostellaria heterophylla (PH), a high-value medicinal herb with significant economic and clinical importance, faces quality standardization challenges due to wild resource depletion and inconsistent cultivated materials. To lay the foundation for establishing reliable quality standards, we developed an integrated metabolomics platform combining ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) with feature-based molecular networking (FBMN) for phytochemical profiling. FBMN enabled relatively comprehensive metabolite annotation, identifying cohesive clusters of alkaloids/organic acids/purines via conserved fragmentation pathways, while cyclopeptides formed isolated nodes due to structural heterogeneity. Multivariate statistical analysis (PCA, OPLS-DA) and machine learning (K-means clustering) revealed significant chemometric differences between geo-authentic (Fujian) and other major commercial (Anhui, Guizhou, Jiangsu) samples, which were further validated through reference crude drug-controlled intergroup comparative analysis. Notably, six signature metabolites (pseudostellarins A/F, heterophyllin J, ferulic acid, dihydro ferulic acid, and azelaic acid) exhibited markedly higher concentrations in Fujian PH, serving as robust geographical marker candidates. These results lay the foundation supporting Fujian’s status as a Dao-di region and establish a systematic framework for geographical traceability. This FBMN-integrated metabolomics approach not only offers a viable strategy for quality control of Dao-di herbs but also proposes a transferable analytical paradigm for geo-authenticity assessment of other herbs. Graphical Abstract

The separation and characterization of phosphorothioate (PS) diastereomers in GalNAc-conjugated siRNA presents a significant analytical challenge due to the high number of isomers present. To tackle this challenge, we explore and optimize various separation techniques, including chromatographic methods (ion-pair reversed-phase, anion exchange, and hydrophilic interaction liquid chromatography) and ion mobility mass spectrometry (IMS) to assess their efficacy in diastereomer separation. Our results indicate that AEX provides the highest diastereomer selectivity among the chromatographic techniques, although none achieved complete diastereomer separation for the chosen antisense and sense strand reference compounds. IMS, applied within a fragment-based tandem mass spectrometry approach, allows separation of all diastereomers of the antisense strand and partial resolution of the sense strand in the gas phase. The comparison of relative LC-UV quantification with IMS data reveals a strong correlation and suggests that IMS can effectively characterize diastereomer ratios not only qualitatively but also quantitatively, establishing IMS as a promising complementary analytical technique for diastereomer separation besides more established LC-UV methods. Graphical abstract

Accounts of Chemical ResearchDOI: 10.1021/acs.accounts.5c00532

Light and electron microscopy utilizes interactions of either photons or electrons with matter to create images from cellular to atomic scale. However, these methods are limited in de novo discovery and spatial mapping of unknown biomolecules. Label free methods such as mass spectrometry or sequencing lack live-cell and subcellular context. Here we introduce a new approach, Ballistic Microscopy (BaM), to image cells with physical nanoparticles. We bombard living cells with millions of nanoparticles traveling at 1000 m/s. Each particle passes through cells, piercing and capturing attoliters of cytoplasm on a hydrogel substrate while preserving spatial information (SPLAT-MAP). This "physical image" of a live cell captures a molecular fingerprint of a cell on a hydrogel film that can be processed post-capture via multiple techniques such as TEM, Cryo-EM, mass spectrometry, confocal imaging, and DNA amplification. Using BaM, we discover previously unknown composition of CLIP170 and Tau3R condensates in HEK cells, uncovering Keratin-18 as a structural element. BaM establishes a new paradigm of "physical imaging" with modular readout platform for spatially resolved live sampling across cells, tissues, and organisms.

Publication date: Available online 8 October 2025 Source: Journal of Proteomics Author(s): Julio A. González-Noriega, Martín Valenzuela-Melendres, Adrián Hernández-Mendoza, Humberto Astiazarán-García, Thalia Islava-Lagarda, Orlando Tortoledo-Ortiz, Ana L. De La Garza-Hernández, Samaria L. Gutierrez-Pacheco, Andrés Alvarez-Armenta, Emmanuel Ríos-Castro, José A. Huerta-Ocampo, E. Aída Peña-Ramos

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00238

Publication date: 1 February 2026 Source: Talanta, Volume 298, Part B Author(s): Meng Yu, Chang Xie, Ding Chen, Qitong Xu, Jun Li, Huawei Wang, Surong Mei

Mass Spectrometry Reviews, Volume 44, Issue 6, Page 873-874, November/December 2025.

ABSTRACT Rationale This paper evaluates the use of a plasma source coupled to an LTQ mass spectrometer for the analysis of explosive residues from soil samples, and in the process examines the advantages and limitations of this new ionization source. Methods Samples were solvent extracted, centrifuged, filtered, and injected into the Solid-Phase Microextraction (SPME)-like device that is coupled to the plasma ion source. The ion source is directly coupled to the orifice of the mass spectrometer, which allows for all ions generated to enter the mass spectrometer. Using the nitrate ion formed in the background and the introduction of ammonium chloride solution, the adduct formations with eight explosives (ETN, HMX, PETN, R-Salt, RDX, tetryl, MHN, and NG) were monitored. Results Reagent ion formation under nitrogen and chloride conditions was studied to determine the optimal conditions for generating adducts with explosive analytes. Extracted soil samples with spiked explosives concentrations ranging from 5 to 1600 ng/mL were analyzed and monitored for the relevant nitrate and chloride adduct masses. For the formation of nitrate adducts, humidified nitrogen at 1500 V was determined to be better than dry nitrogen. For the formation of chloride adducts, 500 mM ammonium chloride at 1500 V was determined to be optimal. Conclusions The highest sensitivity can be achieved by directly coupling the plasma source to the orifice of the LTQ mass spectrometer. A maximum of 2 μL of sample can be introduced in this configuration, but the potential exists to modify the technique for larger volumes.

Current methods for protein level quantification in mass spectrometry-based proteomics do not scale with the increasing number of samples because of limited system memory and algorithmic complexities. Here we propose a new data structure that supports parsing of input as data stream, improve state of the art quantitation methods in performance by orders of magnitudes, and provide a generic method to boost the precision and accuracy by fragment weighting.

Journal of Mass Spectrometry, Volume 60, Issue 10, October 2025.

Environmental Science & TechnologyDOI: 10.1021/acs.est.5c11270

J. Anal. At. Spectrom., 2025, Advance Article DOI: 10.1039/D5JA00254K, Paper Open Access &nbsp This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.Shaun T. Lancaster, Ben Russell, Thomas Prohaska, Johanna Irrgeher A nitrous oxide/ammonia gas mixture has been evaluated for interference removal on 10 important radionuclides for nuclear decommissioning. To cite this article before page numbers are assigned, use the DOI form of citation above. The content of this RSS Feed (c) The Royal Society of Chemistry

Synucleinopathies, including Parkinson's disease, are neurodegenerative diseases characterized by intracellular inclusions containing the amyloidogenic protein alpha-synuclein. While classically considered to be brain disorders, increasing evidence suggests involvement of the gut, with alpha-synuclein aggregates potentially propagating to the brain via the vagus nerve. Evidence also suggests that the vermiform appendix is particularly susceptible to alpha-synuclein aggregation, and appendectomy impacts the onset of Parkinson's disease. However, the mechanisms underlying the aggregation of alpha-synuclein in the vermiform appendix remains poorly understood. To explore this, we assessed aggregation properties in postmortem appendix tissues from healthy controls and synucleinopathy patients using the alpha-synuclein seed amplification assay (alpha-synuclein-SAA) and performed total RNA sequencing alongside differential bisulfite-hybridization-based DNA methylation analysis in the same tissues to investigate the molecular underpinnings. Moreover, we determined alpha-synuclein cleavage patterns by cataloging soluble alpha-synuclein proteoforms from postmortem substantia nigra and post-surgical appendix tissues using top-down mass spectrometry (TD-MS). Alpha-synuclein-SAA was positive in appendix samples for 68.75% of synucleinopathy patients and 6.6% of controls. Genomic profiling revealed dysregulated expression of genes linked to protein folding/degradation, immune/inflammatory responses, and ciliary dynamics in synucleinopathy appendix tissues. TD-MS identified 65 distinct alpha-synuclein proteoforms in the substantia nigra and appendix, with 9 unique to the appendix. Further, in silico modeling revealed higher aggregation propensity of alpha-synuclein proteoforms in the appendix versus substantia nigra. Together, our findings suggest that a tissue environment of alpha-synuclein dysproteostasis in the appendix has the potential to contribute to the development of synucleinopathies.

Juice from noni fruit, Morinda citrifolia, has multiple health benefits but the unpleasant odors of raw juice limits consumer adoption. The two-step process of deodorizing noni juice by rotary evaporation (rotovap) followed by mixing with pineapple juice produces a beverage that has a less off-putting odor and is more palatable. Analysis of total soluble solids and titratable acidity confirms that pineapple juice sweetens the taste profile. Sensory evaluation by trained panelists found that 80% pineapple with 20% rotovap (20 RVN) received higher scores for both sweetness and freshness than 80% pineapple and 20% original noni juice (20 OGN) highlighting the impact of noni juice volatiles on these organoleptic qualities. Noni juice volatiles were further investigated using dynamic headspace sampling (HS) coupled to gas chromatography/mass spectrometry (GC-MS) with principal component analysis (PCA) of volatile profiles. These analyses show that total volatiles decreased in rotovap treated juices, pineapple juice and pineapple/noni juice mixtures grouped more closely while untreated (OGN) and the water fraction removed by rotovap were strongly associated. Together these results suggest that treatment by rotovap removes unpleasant odors from noni juice and mixture with pineapple juice produces a sweeter, fresher, more palatable beverage potentially suitable for the consumer market.

Extracellular vesicles (EVs) play a crucial role in cell-cell signaling, and their dysregulation is linked to various pathologies. EVs can originate from the plasma membrane as microvesicles, or form within endosomal compartments that fuse with the plasma membrane, releasing their intraluminal vesicles in the extracellular space as exosomes. However, distinguishing between these two types of EVs is challenging due to their common protein markers, necessitating cumbersome isolation methods. To overcome this limitation, we developed an innovative tool using Rab7-mediated proximity labelling and mass spectrometry. This technological advancement enables the covalent labeling of Rab7 partners allowing the tracking of intracellular and extracellular exosomal cargoes.

The development of insecticide resistance, high cost, misuse, dearth of technical expertise, and restrictive legislation associated with synthetic insecticides have necessitated the development of alternatives. Lessons from plant-insect interactions demonstrate that plant terpenes are worthy probes for insecticidal exploration. Hence, this study screened n-hexane fractions of Zingiber officinale and Moringa oleifera oils as protectant against Callosobruchus chinensis and revealed their chemical profiles using Gas Chromatography Mass Spectrometry (GC-MS). M. oleifera (LC50; 0.007 microlitre) was found to be more toxic than Z. officinale oil (LC50; 0.055 microlitre) to C. chinensis. The oils showed a positive correlation with concentration at 24h (r = 0.959), 48h (r = 0.977), 72h (r = 0.915) and 96h (r = 0.924). GC-MS revealed 21 and 15 volatile compounds in Z. officinale and M. oleifera oils, respectively. The most dominant were 5-(1, 5-dimethyl-4-hexenyl)-2-methyl-1,3-Cyclohexadiene (13.64%) and 8-Octadecenoic acid, methyl ester (34.52%) in Z. officinale and M. oleifera oils, respectively. The plant fractions reduced the oviposition potential, egg hatching rate, and adult emergence of C. chinensis. Taken together, the results demonstrate possible developmental and inhibitory effects of the oils against C. chinensis and points to its possible inclusion in Integrated Pest Management (IPM) practices for C. chinensis.

Antileukemia immunity is essential for disease control and maintaining treatment-free remission in chronic myeloid leukemia (CML). Immunotherapeutic approaches hold significant promise for enhancing immune-mediated disease control. However, successful immunotherapy relies on identifying CML-associated antigens and achieving robust antigen presentation. Protein kinases are central regulators of signaling, and protein turnover, influencing which peptides are processed and presented on HLA molecules. Indeed, previous works with CDK4/6 or MEK inhibitors showed that kinases influence immune recognition of cancer cells. However, the molecular mechanisms underlying the modulation of the immunopeptidome by kinases inhibitors treatment remained unclear. Here, we investigated whether pharmacological inhibition of key kinases, including BCR-ABL, JNK and LCK, could be leveraged to reshape the immunopeptidome of CML cells, enhancing the exposure of specific tumor-associated antigens. By employing high-sensitive mass spectrometry (MS)-based immunopeptidomics, we quantified more than 20,000 HLA-I peptides. Pharmacological suppression of LCK, JNK and BCR-ABL drastically remodels the antigen landscape, impacting the exposure of about 4,000 HLA-I ligands. By integrating immunopeptidomics, phospho-immunopeptidomics, and proteomic, our study revealed three complementary molecular mechanisms of kinase-dependent antigen control: direct phosphorylation of HLA-I peptides (i), modulation of source protein stability (ii), and control of transcription factors governing antigen expression (iii). Moreover, comparative HLA ligandome analysis of benign hematological specimens and CML primary samples identified a panel of about 90 frequently presented CML-exclusive peptides, whose presentation can be significantly increased by LCK and JNK inhibition. Our study establishes a framework to rationally combine kinase inhibitors with immunotherapies to enhance antigen visibility and improve antileukemic immunity.

The global spread of carbapenemase-producing Klebsiella pneumoniae represents a critical threat to public health, necessitating the development of rapid, sensitive, and reliable diagnostic methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has gained attention as a high-throughput, rapid diagnostic tool for detecting carbapenemase-mediated hydrolysis. However, the effectiveness of conventional organic matrices is limited by inadequate stability, low detection sensitivity, and substantial background interference. In this study, we developed novel bimetallic nanotubes (N-CNT@Zn-Ga/MOF@Au) as a MALDI matrix by integrating nitrogen-doped carbon nanotubes (N-CNT) with a Zn-Ga-based metal–organic framework (MOF) and gold nanoparticles (AuNPs). This nanocomposite exhibited a high surface area, enhanced light absorption, and reduced background noise, contributing to improved signal intensity, reproducibility, and analytical sensitivity. Leveraging this matrix, we analyzed 204 K. pneumoniae clinical isolates for carbapenemase activity through imipenem hydrolysis assays. The method demonstrated exceptional performance, achieving 97.2% sensitivity and 100% specificity within 15 min, with KPC-2-producing strains detected with 100% accuracy. Extending incubation to 1 h allowed for reliable detection of all carbapenemase types, including weaker enzymes such as OXA-48. These findings demonstrate the potential of N-CNT@Zn-Ga/MOF@Au as an advanced MALDI matrix for rapid and accurate detection of carbapenemase activity, with important applications in resistance monitoring and clinical decision support. Graphical Abstract

Mouse vomeronasal sensory neurons are continuously generated from stem cells and differentiate to express V1R or V2R G-protein coupled receptors (GPCRs), along with their respective Gi2 or Go G-protein subunits. We have previously reported that Go-type neurons exhibit elevated expression of endoplasmic reticulum (ER) chaperones and a distinctive hypertrophic, gyroid ER architecture. Here we identify full-length mouse Cnpy1 with its expression and localization exclusive to the ER of Go neurons. Cnpy1 deletion resulted in mice that were deficient in Go neuronal activation upon exposure to vomeronasal stimuli and a marked reduction in male-male aggressive behavior. In Cnpy1-/- mice, Go neuron develop normally till birth, but undergo selective, progressive apoptosis during postnatal development, despite normal trafficking of V2R GPCRs to dendritic tips. Immunoprecipitation and mass spectrometry revealed that Cnpy1 associates with V2R GPCRs and other ER chaperones. Together, these findings identify Cnpy1 as a component of an ER chaperone complex essential for Go neuron signaling and survival.

Publication date: Available online 7 October 2025 Source: International Journal of Mass Spectrometry Author(s): Eugene Moskovets

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00266

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00273

Proteases play critical roles in many biological processes and diseases. Proteases tend to have limited and overlapping specificities, which hinders our understanding of their roles in physiology. We designed a naive protease substrate library with the aim of fully utilizing modern mass spectrometry proteomics tools to quantitatively map protease substrate specificity. A library of approximately 200,000 peptides was efficiently produced in E. coli with sufficient library diversity to identify cleavage efficiency of thousands of substrates for most proteases in a single assay. Each experiment results in ample substrate kinetics data to build a positional specificity matrix that quantitatively maps protease specificity. We apply this method to two different proteases, GluC commonly used as a proteomics tool, and Fibroblast Activation Protein Alpha that is overexpressed in activated fibroblasts of epithelial cancers and fibrotic diseases. This research quantitatively maps substrate specificity for GluC and FAP in a single assay and can be applied to map substrate specificity for most proteases.

Radiation exposure from radiological or nuclear events, medical treatments, or spaceflight poses significant health risks, yet human-specific models to investigate radiation effects on skin remain limited. This study establishes a novel in vitro platform using a full-thickness bioengineered human skin equivalent colonized with natural mixed human microbiota (coHSEs) to assess radiation-induced biological responses. We exposed coHSEs to acute doses of up to 4 Gy with x-rays and evaluated their viability, structural integrity, and molecular responses over 25 days. The coHSE model demonstrated sustained viability without dose-dependent opportunistic microbial overgrowth when procedural optimizations were applied. Radiation-induced epidermal remodeling did not compromise tissue architecture or swabbing-based sample collection. Cell proliferation analyses revealed dose- and time-dependent dynamics, with consistent dermal cell density maintained across radiation doses. Comparative multi-omic analyses, including untargeted metabolomics, targeted lipidomics, and 16 S metagenomics, revealed conserved metabolic and microbial responses to radiation in both coHSEs and skin from irradiated mice. Enriched pathways such as arachidonic acid and fatty acid metabolism, along with shifts in microbial taxa including Lachnospiraceae, support the translational relevance of the coHSE model. This system offers a scalable, ethical, and physiologically relevant platform for radiation biology, biodosimetry, and therapeutic development, advancing terrestrial health research with promising application for space research.