Understanding drug targets and their effects in living cells is challenging. Here, the authors develop SICFA, a label-free proteomics method that sensitively detects drug-induced protein stability changes, thereby identifying targets and downstream pathways directly in living cells.

RNA-protein complexes are critical factors in development, homeostasis, and disease. RNA proteomics methods are essential for characterizing these complexes but suffer from high levels of background, which hinders identification of ribonucleoprotein (RNP) components. Here, we present RNA Antisense Purification followed by Mass Spectrometry 2.0 (RAP-MS 2.0), an updated version our original RAP-MS protocol with innovations in bead preparation, RNA capture, and peptide purification. RAP-MS 2.0 has lower background than our original protocol, and allows lysate to be reused to capture multiple RNAs. We demonstrate that RAP-MS 2.0 recapitulates known RNPs for 7SL, 7SK, RMRP, U1, U2, U6, U7, and Xist. Additionally, we use RAP-MS 2.0 to identify novel RNA-protein interactions between Xist and TREX components and U1 with FET family transcriptional regulators.

Journal of Proteome ResearchDOI: 10.1021/acs.jproteome.5c00296

Publication date: Available online 24 November 2025 Source: Journal of Proteomics Author(s): Isabela de Oliveira Cavalcante Pimentel, Wellington da Silva Santos, Fabio Montoni, Alison Felipe Alencar Chaves, Rosangela Aparecida dos Santos Eichler, Ismael Feitosa Lima, Talita Souza-Siqueira, Milton Yutaka Nishiyama, Lilian de Jesus Oliveira, Emer Suavinho Ferro, Leo Kei Iwai

Journal of the American Society for Mass SpectrometryDOI: 10.1021/jasms.5c00172

Analytical ChemistryDOI: 10.1021/acs.analchem.5c05874

Publication date: Available online 25 November 2025 Source: Journal of Chromatography A Author(s): Muhamad Yahia Kazmouz, Mátyás Mayer, Attila Felinger

This study presents the development of several approaches to obtain, for the first time, proteins and bioactive peptides from grapefruit peels. The experimental conditions of three extraction methodologies based on the use of Ultrasound-Assisted Extraction (UAE) with Tris-HCl buffer, UAE with Natural Deep Eutectic Solvents, and Pressurized Liquid Extraction (PLE) were optimized. Among them, PLE proved to be the most effective approach, yielding a protein-rich extract containing 70% protein. Protein extracts obtained by PLE were submitted to enzymatic hydrolysis with Thermolysin, Alcalase, and Alcalase Pure, and the resulting protein hydrolysates were then evaluated for their antioxidant and antihypertensive activities. Finally, peptides and other molecules present in the protein hydrolysates were identified using ultra-high liquid chromatography coupled to hybrid quadrupole-Orbitrap mass spectrometry. Thus, a total of 75 peptides along with 31 additional compounds mainly classified into the families of flavonoids, carboxylic acids, amino acids, and carbohydrates were identified. Graphical abstract

Publication date: 1 March 2026 Source: Talanta, Volume 299 Author(s): Ludovica Sofia Guadalupi, Cosima Damiana Calvano, Tommaso R.I. Cataldi, Andrea Bernardi, Luca Magazzini, Luciano Tadiello, Valeria Cipolletti, Marco Arimondi

Publication date: Available online 24 November 2025 Source: Analytica Chimica Acta Author(s): Amirsalar Mansouri, Nipun Babu Varukattu, Brennan J. Curole, Omeed Moaven, Jiri Adamec

J. Anal. At. Spectrom., 2026, Accepted Manuscript DOI: 10.1039/D5JA00389J, PaperChaofeng Li, Zhu-Yin Chu, Peng Peng Sn is the main isobaric interference for Cd isotopic compositions determination. To eliminate the Sn, two step separation methods using anion resin combined with commercially available TRU and UTEVA extraction... The content of this RSS Feed (c) The Royal Society of Chemistry

ABSTRACT Rationale Inductively coupled plasma (ICP) is a commonly used ion source for mass spectrometry-based chemical analysis of a wide range of materials. Traditional ICP ion sources use high power (> 1000 W) and significant gas flow (> 10 L/min), rendering them unsuitable for spaceflight, as they are too resource-intensive for planetary spacecraft. Methods To address the technology gap, we designed and developed a laser ablation microwave-induced plasma mass spectrometer (LA-MIP-MS) and experimentally validated the analytical performance of a prototype instrument capable of providing in situ analyses during planetary science missions. We developed a low-pressure plasma ion source and interfaced it to a heritage quadrupole mass spectrometer (QMS) to perform elemental and isotopic analysis of solid samples via laser ablation. The low power plasma ion source was generated at

Researchers present GlycoGenius, an open-source tool that automates complex glycomics data analysis. It streamlines workflows, identifies known and previously unreported glycans, and enables faster, more accessible insights into glycobiology.

Journal of Proteome ResearchDOI: 10.1021/acs.jproteome.5c00721

Background: Invasive diagnostic techniques for pleural mesothelioma (PM) delay early detection, underscoring the need for non-invasive biomarkers. Exhaled breath condensate (EBC) is a promising matrix but has extremely low protein concentration. This study applies an amphipol (APol)-based concentration method to enhance detection of potential PM biomarkers in the EBC proteome. Methods: Four replicate EBC samples from a healthy individual were processed using APols or lyophilization. Subsequently, 145 EBC samples from PM patients (n=25), lung cancer patients (n=28), asymptomatic asbestos-exposed individuals (n=60) or healthy controls (n=32) were concentrated with APols and analysed by liquid chromatography coupled with tandem mass spectrometry. Technical variability from batch effects and collection devices was addressed using omicsGMF, for simultaneous imputation and covariates correction. Statistical analysis employed linear models with technical and clinical covariates. Results: APols outperformed lyophilization, yielding 1.7-fold higher proteome coverage and improved reproducibility. Although high data missingness was observed, proteome analysis across 145 samples identified 248 unique proteins. OmicsGMF corrected the substantial variability, while maintaining biological signal. Whereas unsupervised clustering did not reveal distinct disease-specific patterns, supervised statistical analysis detected overall subtle differences between clinical groups (PERMANOVA p-value = 0.008), though no individual proteins reached statistical significance after adjustment for technical and clinical covariates and correction for multiple testing. Conclusion: APols outperforms lyophilization for concentrating proteins in low-abundant EBC samples, enabling enhanced proteome coverage for biomarker discovery studies. However, the inherent high variability of EBC proteomes has a strong effect on detecting possible biomarkers across clinical groups.

Protein isoform inference from bottom-up mass spectrometry (MS) relies on database search strategies that assume the reference protein database accurately reflects the full repertoire of genetic and transcriptomic states present in the sample being analyzed. Long-read RNA sequencing (lrRNA-seq) now enables simultaneous recovery of complete transcript structures and the genetic variants present on each molecule, offering a direct route to allele-specific isoforms, yet this capability has not been fully leveraged to improve MS-based proteogenomics workflows. Here, we develop an end-to-end workflow for constructing and searching haplotype-resolved, sample-specific proteomes using matched lrRNA-seq and MS data. We benchmark multiple phasing algorithms on PacBio lrRNA-seq from Genome-in-a-Bottle samples and identify methods that achieve high phasing accuracy and completeness on transcriptomic reads. Our open-source, modular Snakemake pipeline performs variant calling, read-based phasing, isoform discovery, haplotype-resolved proteome construction, MS search, and downstream annotation. To demonstrate its utility, we apply the workflow to an induced pluripotent stem cell line (WTC11) and to an osteoblast differentiation time course, showing that haplotype-resolved databases enable detection of variant and splice peptides, allele-specific protein isoforms, and linked variants not detectable with reference-only proteomes. Together, our results demonstrate that lrRNA-seq-based phasing is feasible and effective for proteogenomics and provide a practical framework for allele-resolved proteome characterization in dynamic or disease-relevant settings.

Anal. Methods, 2025, Advance Article DOI: 10.1039/D5AY01659B, Technical NoteShin Nishiumi, Tomonori Yokoyama, Noriyuki Ojima Mass spectrometry, including GC/MS, LC/MS, and CE/MS, can analyze many different molecules in a single measurement, facilitating metabolome analysis. 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

Hypothyroidism is a prevalent endocrine disorder characterized by insufficient thyroid hormone (T3T4) production. Thyroglobulin (Tg) serves as the prohormone for T3 and T4 production, with many variants of uncertain clinical significance due to genetic diversity in the Tg gene. We leveraged the large-scale All of Us biobank to investigate the disease association of prevalent yet undercharacterized Tg variants. We related variant presence to thyroid-stimulating hormone levels and levothyroxine (LT4) usage as proxies for thyroid function. This identified R152H, Q870H, A993T, P1012L, and P1494L variants linked to increased LT4 usage and decreased thyroid function, while the R320C variant was associated with decreased thyroid function. Molecular characterization in Fisher rat thyroid cells revealed decreased secretion efficiency of R152H, Q870H, and R320C variants. Affinity purification-mass spectrometry demonstrated that secretion-deficient variants showed higher engagement with the protein homeostasis network, indicating protein quality control defects as the pathophysiology mechanism. In contrast, secretion-competent A993T and P1494L variants showed elevated interactions with degradation and antigen-presentation pathways, suggesting an alternative pathophysiology possibly linked to Hashimoto disease, an autoimmune condition with overproduction of autoantibodies that target thyroid proteins. In support, participants carrying the A993T or P1494L variants had elevated anti-TPO antibody levels. We estimate ~150,000 US individuals currently taking levothyroxine could benefit from precision medicine targeting these variants, with ~100,000 carrying Q870H. Our findings highlight the power of combining large public biobank data with molecular characterization to understand Tg genotype-to-phenotype relationships. Q870H represents a candidate for molecular therapies to restore secretion, offering precision medicine beyond LT4 replacement therapy.

Journal of Mass Spectrometry, Volume 60, Issue 12, December 2025.

Non-target screening (NTS) using liquid chromatography-high-resolution mass spectrometry has become essential for uncovering unknown contaminants in complex matrices such as industrial wastewater. A key goal in these applications is detecting and interpreting time trends, especially spill-related events. The clarity of multivariate models depends on the quality of feature lists from various software tools. In this study, we evaluated five peak picking tools (MarkerView, MZmine3, XCMS, OpenMS, and SIRIUS) for unsupervised time trend exploration using sparse principal component analysis (SPCA). SPCA selects the most informative features per component, improving interpretability and reducing confounding variables. Two datasets were used: a controlled validation set of pooled wastewater samples with spiked target compounds exhibiting known profiles and a real-world dataset comprising 52 consecutive daily industrial wastewater samples. The first dataset facilitated analysis of tuning parameters with SPCA distinguishing spiking patterns associated with components, highlighting differences in feature/artifact prioritization across tools. Tools XCMS, MZmine3, and OpenMS showed higher consistency and were selected for next analysis. In the second phase, SPCA was combined with stratified bootstrapping (SBS-SPCA) to assess the reliability of trend detection, exemplified by specific targets. Five out of nine markers, showing more temporal persistency, were robustly detected across tools (selection frequency > 70%) under optimized tuning conditions. These findings indicate that interpretable, sparse models enhance marker detection in unsupervised settings and shed light on how software-driven feature structures impact multivariate outcomes in time-series NTS data. Such insights are especially pertinent for future high-throughput applications involving temporally dynamic exposure scenarios. Graphical abstract

Chemistry – A European Journal, EarlyView.

Nitrosamines are classified as carcinogenic chemicals and are a growing public health concern. They have been widely documented in food, which is the main route of human exposure. However, there is little research on their appearance in biological samples, such as feces. This study presents a methodology for determining the concentration of 14 nitrosamines in cooked hams and fecal samples obtained from animals fed with them, allowing changes in the nitrosamine concentration during digestion to be identified. The novelty of this research lies in the fact that it is the first to evaluate whether incorporating polyphenols into cooked ham influences the levels of NAs in feces using an animal model. The proposed analytical method based on ultrasound-assisted extraction of the nitrosamine high-performance liquid chromatography and mass spectrometry using a triple quadrupole achieved low limits of quantification (in the 0.44–47 ng g−1 range), good precision (coefficient of variation of less than 12.4%), and trueness for both matrices. A total of 68 samples were analyzed, with five nitrosamines detected in the cooked ham samples and seven in the fecal samples. Statistical analysis revealed that the nitrosamine profile mainly depended on the biological matrix. While the preservative did not significantly affect the nitrosamine profile in cooked ham, it did modulate the nitrosamine concentration in feces. Specifically, diets containing polyphenols significantly reduced fecal nitrosamine levels compared to diets containing nitrites, suggesting a possible protective role against the formation of these compounds in the gut. Graphical Abstract

A review of recent innovations driving low-input phosphoproteomics, focusing on miniaturized workflows, multiplexed labelling, automated enrichment, and advanced acquisition methods for scarce biological material.

Silver carp (Hypophthalmichthys molitrix) has been identified as one of the four famous Chinese carps. The Yangtze River is the main home to silver carp. However, during the “ten-year fishing ban”, illegal fishing frequently occurred, law enforcement agencies failing to discriminate between farmed and wild fish from the Yangtze River. Therefore, there is a strong need to develop a simple and effective method to discriminate between farmed and wild silver carp from the Yangtze River. In this study, 76 fatty acids were analyzed in 266 silver carp samples. The top 8 fatty acids that exhibited the best performance were identified as candidate biomarkers. The different origins of wild silver carp were also identified. Based on receiver operating characteristic (ROC) curve analysis, myristic acid proportion of total fatty acids was considered as a biomarker which has high sensitivity and specificity in discrimination of farmed and wild silver carp.

Publication date: Available online 22 November 2025 Source: Journal of Chromatography B Author(s): Lisbet Sørensen, Mari Egeness Creese, Marie Ibrekk, Rikke Torvanger, Raymond Nepstad, Julie Metzger, Trond R. Størseth, Julia Farkas, Bjørn Henrik Hansen