PIWI-interacting RNAs (piRNAs) are critical for transposon silencing and genome integrity, as well as gene expression regulation and antiviral immunity in metazoans, yet the molecular mechanisms governing their biogenesis remain incompletely understood. The participation of the splicing-associated process in piRNA biogenesis has been emphasized in multiple species, but the key factors and mechanisms remain elusive. Here, we identified SUGP1 (SURP and G-Patch Domain Containing 1) in BmE (a unique model cell system with a complete piRNA biogenesis pathway) as a key splicing factor that functions in piRNA biogenesis. Through CRISPR-Cas9-mediated gene knockdown in cultured cells combined with RNA-seq, small RNA-seq, and IP-mass spectrometry (IP-MS), our results reveal that SUGP1 deficiency disrupts piRNA accumulation, alters mature piRNA length distributions, and activates transposon expression. Immunofluorescence and Western blot (WB) analyses further demonstrate that SUGP1 interacts with Y-box protein (YBP), which is key regulators of RNA metabolism. Functional validation in Drosophila SUGP1-RNAi lines highlights evolutionary conserved and species-specific roles of SUGP1 in piRNA maturation. Collectively, our data uncover a dual role for silkworm SUGP1 in coordinating YBP-dependent piRNA biogenesis, thus elucidating a novel mechanistic framework for piRNA pathway regulation. Our work also underscores the silkworm as a unique model for studying non-canonical piRNA biogenesis mechanisms, with implications for treating transposon dysregulation-linked diseases.

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00324

Publication date: Available online 9 October 2025 Source: Journal of Chromatography A Author(s): Rui Wang, Wei Wei, Man Yang, Yang Liu, Xue Meng, Shiming Fang, Jin Li, Kunze Du, Yanxu Chang

Publication date: Available online 8 October 2025 Source: Journal of Chromatography A Author(s): Hira Zulfiqar, Maria Llompart, Natalia Montero, Andres Duque-Villaverde, Daniele Fabbri

Protein identification by mass spectrometry is a pivotal step in proteomics. Numerous methods have been developed to securely and effectively identify proteins derived from experimentally detected peptides by mass spectrometry. The dominant approach is based on the assumption that each experimentally identified peptide can be matched with a peptide included in a database of peptide sequences, generated by in silico digestion of proteins with a specific proteolytic enzyme. In this way, the protein containing the peptide can be identified. In a more advanced approach, the proteins and their in silico digested peptides in the database are transformed into theoretical mass spectrometry spectra, and search engines match the experimentally obtained spectra to these theoretical spectra generated from protein and peptide sequences. We developed an alternative method for protein identification using Core Unique Peptides (CrUPs) and the Uniquome, termed as Uniquome-Based Protein Identification Method (UB-PIM). According to this method, instead of searching for peptides in the database of in silico_digested peptides, we search for CrUPs within the experimentally obtained peptides by mass spectrometry. If a peptide contains at least one CrUP, it can be directly correlated to the protein from which the CrUP is derived. Because of the unique nature of CrUPs, peptides obtained by MS can securely and uniquely identify the protein of origin. This provides a reference space in which even single-peptide identifications can achieve high specificity, reducing the ambiguity caused by shared or homologous sequences and improving the interpretability of MS data. Furthermore, UB-PIM can be applied to any type of peptide and is effective with both Data-Independent Acquisition (DIA) and Data-Dependent Acquisition (DDA) approaches, as well as with top-down and bottom-up proteomics. This allows confident protein identification from minimal evidence, expands the scope of detectable proteins, and remains computationally efficient, rapid, and universally applicable.

Halogenated natural products (HNPs) are a diverse group of organohalogen compounds, predominantly found in the marine environment. Selected HNPs show structural similarity to persistent organic pollutants (POPs), but data on their occurrence is limited. An analytical method for the detection of 17 commonly reported HNPs, as well as selected POPs, using gas chromatography with electron-capture negative ionisation mass spectrometry (GC/ECNI-MS) was established and validated for salmon and blue mussel. The validation confirmed the applicability of the method with recovery rates and inter-day coefficients of variations of 52–101% and 3.7–16%, respectively. The subsequent application of the method to 17 samples of frequently consumed seafood products from the German retail market demonstrated its suitability for a wide variety of matrices. HNPs were detected in all analysed samples, with contents ranging from 

J. Anal. At. Spectrom., 2025, Accepted Manuscript DOI: 10.1039/D5JA00232J, PaperPierre-Emmanuel Peyneau, Léonard Seydoux, Mickael Tharaud Single particle inductively coupled plasma-mass spectrometry (sp-ICP-MS) produces time series - time scans - that require processing to extract meaningful information regarding the nanoparticles that are analysed. In this work,... The content of this RSS Feed (c) The Royal Society of Chemistry

J. Anal. At. Spectrom., 2025, Accepted Manuscript DOI: 10.1039/D5JA00309A, Critical ReviewChufan Zhou, Qiang Huang, Minming Cui, Xun Wang, Xinbin Feng The rapid advancement of artificial intelligence (AI), particularly machine learning (ML), is revolutionizing stable isotope geochemistry, enhancing mass spectrometry-based analytical techniques and unlocking transformative capabilities in data interpretation and geochemical... The content of this RSS Feed (c) The Royal Society of Chemistry

J. Anal. At. Spectrom., 2025, Advance Article DOI: 10.1039/D5JA90048D, Atomic Spectrometry UpdateAyush Agarwal, Eduardo Bolea-Fernandez, Robert Clough, Andy Fisher, Bridget Gibson, Steve Hill This review covers advances in the analysis of advanced materials, metals, fuels and lubricants, nanostructures, ceramics, refractories, organic and inorganic chemicals, catalysts and nuclear materials by a range of techniques including X-ray, ICP, LIBS, mass spectrometry, synchrotron-based techniques, plus non-destructive and ablation surface techniques. 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

Background and Aim: Indigenous medical systems employ unique pharmaceutical techniques to meet the therapeutic needs. In the Indian System of Medicine, Ayurveda, a unique method called Ayolepam is described that facilitates efficient iron absorption from its source to the formulation. Punarnavayolepa Choornam (PC), prepared from Boerhavia diffusa L., is one such novel Ayolepam formulation developed by a traditional Ayurveda school of South India, widely used in the clinical management of iron deficiency anaemia. This study aims at scientifically evaluating the Ayolepam technique by assessing the iron binding and bioavailability properties of PC, prepared from the leaves, root and whole plant of B. diffusa, using in-vitro model systems.. Experimental Procedure: The iron content in both raw and processed samples of PC was quantified using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A simulated in-vitro digestion model employed to assess the release of bioavailable iron from the formulation. Subsequently, iron bioavailability was evaluated using the Caco-2 cell model of human intestinal epithelium following the ferrozine method. Results and Conclusion: PC preparation from leaves, whole plant and roots of B. diffusa, showed significant increase in iron content compared to the raw material. In-vitro digestion studies confirmed the efficient release of bioavailable iron from these formulations, and subsequent Caco-2 cell assays confirmed their iron bioavailability properties. In conclusion, these findings provide a preliminary scientific basis for this novel and unique pharmaceutical design from a traditional school of Ayurveda, supporting its clinical application.

Publication date: Available online 8 October 2025 Source: Journal of Proteomics Author(s): Celso Vitor A.Q. Calomeno, Hulyana Brum, Rodrigo S.C. Brant, Marlon D.M. Santos, Luis Miguel Muñoz-Gómez, Ana Gisele da Costa Neves-Ferreira, Richard Hemmi Valente, Michel Batista, Paulo C. Carvalho

ABSTRACT Rationale Viticis fructus (VF) is a valuable traditional Chinese herbal medicine with high commercial value. In the Chinese Pharmacopoeia (Ch.P., 2020 edition), the quality of VF is mainly evaluated by its casticin content. However, varying growth and production conditions, such as cultivation environment, origin, and harvesting process, result in highly variable yields, quality, and composition. The aim of this study was to propose a new method for discriminating and quantifying VF from different regions. Methods The chemical constituents of VF from different regions were collected by ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) using electrospray ionization (ESI) in both positive and negative ion modes, and the chemical constituents were qualitatively analyzed by Waters UNIFI portal 1.9.4.0 software combined with the self-developed database of VF. The spectra obtained by Progenesis QI 27.26.1020 and EZinfo 2.0 software were processed by peak standardization, peak extraction, peak alignment, peak matching, and so forth, and the information of compound retention time and mass–charge ratio was obtained. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed by SIMCA-P 14.0 software to study the different components of VF from different regions. Results A total of 102 components were identified from VF. The results of PCA and OPLS-DA showed that there were significant differences in the chemical components of VF from different regions. Twenty-seven different components were obtained by variable importance for the projection (VIP) > 1 screening, including 12 terpenoids, 7 phenylpropyl compounds, 3 phenolic acids, 2 flavonoids, 2 fatty acids, and 1 steroid. Conclusions By establishing a method of UPLC-QTOF-MS combined with multivariate statistical analyses, this study clarifies the difference in chemical components between VF from different regions, which provides a basis for the quality control of VF and provides basic information for revealing the impact of the ecological environment on the accumulation of compounds in VF.

Journal of Mass Spectrometry, Volume 60, Issue 11, November 2025.

Journal of Mass Spectrometry, Volume 60, Issue 11, November 2025.

Journal of Mass Spectrometry, Volume 60, Issue 11, November 2025.

RNA Polymerase II (Pol II) transcription is highly regulated at two early steps in the transcription cycle: Pre-Initiation Complex (PIC) assembly with its coupled initiation, and promoter-proximal pausing with its controlled release. Here, we developed an optimized biochemical purification method that captures endogenously tagged chromatin-bound Pol II complexes under native conditions at these rate-limiting steps. We then identified a large set of Pol II interactors by mass spectrometry and determined the footprints of these assemblies on promoters with high resolution. Many well-known and new or understudied factors were identified as associated with the PIC and promoter-proximal paused complexes, indicating that despite decades of efforts, these rate-limiting steps of the transcription cycle are far from being completely understood. The new and understudied factors implicate novel mechanisms of regulation that will need to be characterized to fully understand Pol II regulation.

Systemic amyloidosis is a potentially fatal protein misfolding disorder usually underdiagnosed in low- and middle-income countries, where limited awareness and restricted access to diagnostic tools contribute ...

Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. Mass spectrometry-based proteomics has allowed proteome-scale quantification of hundreds of thousands of ubiquitination sites (ubi-sites), however the functional importance and regulatory roles of most ubi-sites remain undefined. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. We identified a core subset of ubi-sites under evolutionary constraint through alignment of ubiquitin proteomics data from six non-human species, and determined ultra-conserved ubi-sites recurring at regulatory hotspots within protein domains. Perturbation proteomics revealed that these highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize functional ubi-sites with roles in cellular signaling, we constructed a functional score for more than 100,000 ubi-sites by integrating evolutionary, proteomic, and structural features using machine learning. Our score identifies ubi-sites regulating diverse protein functions and rationalizes mechanisms of genetic disease. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying protein ubiquitination.

Chitinase-3-like protein 1 (CHI3L1) is a multifunctional extracellular glycoprotein implicated in tumor progression, immune suppression, and fibrosis, making it an attractive but challenging therapeutic target. To explore its chemical tractability, we applied an affinity selection-mass spectrometry (AS-MS) workflow to screen 10,000 small molecules for CHI3L1 binding. The screen yielded 124 initial hits with a hit rate of 1.24%, which were prioritized based on chemical suitability, and six candidates were advanced for validation using microscale thermophoresis (MST). Among these, compound A9 exhibited a clear, dose-dependent binding response in MST with a Kd of 182 M. Molecular docking supported these findings, revealing that A9 forms hydrophobic and hydrogen-bonding interactions within a defined pocket of the CHI3L1 structure. Although modest in affinity, A9 represents the first small molecule binder of CHI3L1 identified through AS-MS. This study provides a proof-of-concept demonstration that CHI3L1 can be chemically engaged using AS-MS, establishing a foundation for future medicinal chemistry optimization and the development of chemical probes targeting this previously undruggable extracellular protein.

Photosynthesis, which sustains life on Earth, depends on highly organized yet adaptable protein assemblies embedded in specialized membranes called thylakoids. Understanding how protein complexes dynamically interact within functional photosynthetic membranes is critical for elucidating cellular energetic metabolism. Here, we present an improved crosslinking mass spectrometry (XL-MS) strategy that captures native protein interactions in functional, photosynthetically active thylakoid membranes from Arabidopsis and Spinach. By monitoring photo-physiology parameters, we demonstrate that electron transport remains physiologically active during the crosslinking process, enabling structural interrogation without disrupting native organization. Mapping crosslinks onto known structures confirms the structural integrity of major complexes and highlights previously uncharacterized assemblies. Our findings pave the way for exploring membrane protein networks in situ and set the stage for integrative structural studies of photosynthetic regulation and adaptation.

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