bims-mascan 2026-01-11 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2026–01–11
sixteen papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

GlypPRM: An Automated Analyzer and Quantification Tool for Glycopeptides Parallel Reaction Monitoring.
RP-UHPLC/MS/MS provides enhanced lipidomic profiling of human serum in pancreatic cancer.
NITCD: A streamlined metabolomics strategy based on bromine isotopes and MS-TDF software.
Enhancing Sensitivity in Targeted Single-Cell Proteomics by Coupling a Dual Ion Funnel Interface with Triple Quadrupole Mass Spectrometer.
Mass Spectrometry Quantification of Epigenetic Changes: A Scoping Review for Cancer and Beyond.
Protocol for stable isotopic tracing to assess cellular lipogenic activity in induced neural stem cells.
An Overview of Liquid Chromatography-Mass Spectrometry (LC-MS) Methods for the Quantification of Antibody-Drug Conjugates.
Sample preparation of Drosophila melanogaster larvae for MALDI imaging.
Nutrient requirements of organ-specific metastasis in breast cancer.
Emerging analytical techniques for lipid profiling in food products: insights into processing effects and quality control.
Improved surfactant-assisted one-pot sample preparation for robust convenient single cell proteomics.
Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2).
BACE2 tunes lipid uptake through lipid transporters shedding supporting cancer cell proliferation.
Protocol for determining gut microbiota metabolites as substrates in mouse metabolism.
13C-Isotope Tracing Structural Lipidomics for Resolving Phospholipid Metabolism Dynamics in Human Breast Cancer Cells.
Quantitative GC-MS/MS in the clinical analysis of eicosanoids: A critical review and discussion, a 40-years historical retrospect, and possible implications for LC-MS/MS.

Anal Chem. 2026 Jan 03.

GlypPRM: An Automated Analyzer and Quantification Tool for Glycopeptides Parallel Reaction Monitoring.

Oluwatosin Daramola, Sherifdeen Onigbinde, Moyinoluwa Adeniyi, Cristian D Gutierrez-Reyes, Mojibola Fowowe, Vishal Sandilya, Yehia Mechref.

Glycosylation is a prevalent and structurally complex post-translational modification implicated in diverse biological processes and diseases. Mass spectrometry (MS)-based glycoproteomics, especially parallel reaction monitoring (PRM), offers high specificity and quantitative power for glycopeptide analysis. PRM enables full MS/MS acquisition for targeted precursors, enhancing signal-to-noise ratios and structural confidence, key advantages over conventional targeted methods. However, the identification and quantification of glycopeptides from PRM data remain challenging due to extensive glycan heterogeneity, site multiplicity, and complex fragmentation patterns. Existing software platforms often lack tailored support for glycopeptide-specific fragmentation logic, glycan structure modeling, or automated spectral interpretation, leaving much PRM-based glycoproteomics reliant on manual workflows. To address these limitations, we developed GlypPRM, a Python-based, fully integrated platform for automated glycopeptide PRM data analysis. GlypPRM supports compositional glycan structure modeling for theoretical fragment ion generation, spectral matching, chromatographic integration, and quantitative analysis for both N- and O-glycopeptides. We validated its performance using glycopeptides derived from bovine fetuin and human serum samples, demonstrating high structural accuracy, reproducibility, and interpretability. GlypPRM also includes advanced visualization, flexible input handling, ion filtering, and publication-ready export formats. This scalable, glycan- and peptide-aware platform establishes a strong foundation for high-confidence PRM-based glycoproteomics in biomarker discovery and disease research.

DOI: https://doi.org/10.1021/acs.analchem.5c06005
Anal Chim Acta. 2026 Jan 22. pii: S0003-2670(25)01346-7. [Epub ahead of print]1384 344952

RP-UHPLC/MS/MS provides enhanced lipidomic profiling of human serum in pancreatic cancer.

Zuzana Lásko, Ondřej Peterka, Robert Jirásko, Anna Taylor, Tomáš Hájek, Beatrice Mohelníková-Duchoňová, Martin Loveček, Bohuslav Melichar, Michal Holčapek.

   BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, mainly due to the late diagnosis and the lack of reliable biomarkers. Lipidomics provides a promising approach for identifying disease-related alterations, but existing methods are often limited to lipid class profiles with insufficient molecular detail. Reversed-phase ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (RP-UHPLC/MS/MS) offers the possibility to determine lipids at the fatty acyl/alkyl level. Here, we address the need for a validated quantitative workflow that enables accurate and reproducible lipidomic profiling of human serum in the context of PDAC.
RESULTS: We developed and validated an RP-UHPLC/MS/MS method using multiple reaction monitoring, enabling the identification of 455 lipid species from 22 subclasses, with 381 species from 21 subclasses quantified. The workflow included a response factor correction for sterol esters, which markedly improved their quantification accuracy. The application to serum samples from 54 PDAC patients and 55 healthy controls yielded highly reproducible data, with clear group separation observed in both unsupervised and supervised statistical analyses. Dysregulation was most prominent in sphingolipids and phospholipids. Very long-chain saturated sphingolipids (≥C22) were downregulated, while some shorter or unsaturated chains showed mild upregulation. Phospholipid alterations were dominated by species containing polyunsaturated fatty acyls, particularly 18:2 and 20:4, with plasmalogens showing the strongest changes. These structurally resolved findings were further supported by gas chromatography - mass spectrometry analysis of fatty acid methyl esters.
SIGNIFICANCE: This validated workflow provides comprehensive quantitative coverage across 21 lipid subclasses with the structural resolution critical for biological interpretation. The detailed mapping of sphingolipid and phospholipid dysregulation in PDAC demonstrates that only the fatty acyl level annotation reveals molecular signatures that may reflect specific enzymatic activities or pathways. The method delivers a robust platform for biomarker discovery and mechanistic studies in cancer lipidomics.

Keywords:  Human serum; Lipidomics; Mass spectrometry; Pancreatic cancer; Reversed-phase liquid chromatography

DOI:  https://doi.org/10.1016/j.aca.2025.344952
Talanta. 2025 Dec 31. pii: S0039-9140(25)01833-8. [Epub ahead of print]301 129342

NITCD: A streamlined metabolomics strategy based on bromine isotopes and MS-TDF software.

Dandan Zhang, Hairong Zhang, Jiajin Yi, Di Lu, Caisheng Wu.

  In metabolomics research, derivatization methods, particularly stable isotope derivatization, are commonly employed to enhance the coverage and qualitative and quantitative accuracy of analyte compounds. In our previous work, we adopted a homolog derivatization approach combined with our in-house developed mass spectrometry triple-dimensional derivatization filter (MS-TDF) software to achieve low-cost metabolomics studies. However, differential derivatization efficiencies across homologs led to an increased false positive rate. In this study, we introduce a natural isotope triple-dimensional combinatorial derivatization (NITCD) strategy that overcomes these limitations. The approach employs 4-bromo-2-hydrazinopyridine, which provides a characteristic 1:1 isotopic doublet pattern (79Br/81Br) for intelligent metabolite identification via MS-TDF software. Combined with 2-hydrazinopyridine as a structurally matched internal standard, the system not only reduces false-positive identifications but also enables reliable relative quantification. This strategy was successfully applied to a metabolomics study on rhein treatment in inflammatory bowel disease (IBD). The experimental results demonstrate that, by using NITCD strategy, 564 target compounds can be detected in mouse plasma, with 148 in colon tissue, and 81 in spleen tissue. More importantly, these metabolites were identified, along with their dynamic changes during rhein treatment. It was found that rhein might reverse the IBD-induced alterations in arachidonic acid metabolism, tyrosine metabolism, primary bile acid biosynthesis, and tryptophan metabolism.

Keywords:  Bromine; Inflammatory bowel disease; MS-TDF; Metabolomics; Rhein

DOI:  https://doi.org/10.1016/j.talanta.2025.129342
bioRxiv. 2026 Jan 02. pii: 2026.01.02.697431. [Epub ahead of print]

Enhancing Sensitivity in Targeted Single-Cell Proteomics by Coupling a Dual Ion Funnel Interface with Triple Quadrupole Mass Spectrometer.

Sehong Min, Pearl Kwantwi-Barima, Issac K Attah, Thomas L Filmore, Matthew J Gaffrey, Reta Birhanu Kitata, Yehia M Ibrahim, Tujin Shi.

Single-cell proteomics (SCP) has emerged as a powerful approach for understanding cellular heterogeneity and biological processes at unprecedented resolution. However, the extremely limited protein content of individual cells (femtogram to picogram levels) pushes current mass spectrometry instrumentation to its sensitivity limits, creating a critical analytical bottleneck. While selected reaction monitoring (SRM) using triple quadrupole (QqQ) instruments offers advantages in sensitivity and reproducibility for targeted proteomics quantification, SRM still struggles with sensitivity for quantification of moderate- or low-abundance proteins from single-cell sample amounts. Here, we report the development and systematic evaluation of a dual ion funnel interface designed to address the sensitivity limitation by significantly enhancing ion transmission efficiency in commercial QqQ mass spectrometers. The dual ion funnel interface, composed of a curved S-funnel followed by a conventional ion funnel, improves ion transmission efficiency while reducing chemical noise through selective ion focusing. The performance of the dual ion funnel interface was systematically compared to standard interface on a TSQ Vantage platform across samples with different levels of complexity. The dual funnel interface demonstrated to provide up to 25-fold improvement in sensitivity across a wide range of protein concentrations in different biological matrices (low complex mouse macrophage and high complex human cells). Critically, enhanced sensitivity was accompanied by increased analytical reproducibility with lower coefficient of variations. Most importantly, the dual funnel interface enabled reliable quantification of low-abundance proteins that were barely detectable or not detected by the standard interface, extending analysis to single-cell equivalent amounts while maintaining excellent reproducibility. These results demonstrate that the dual funnel interface addresses the critical bottleneck in quantitative targeted proteomics, providing a technological foundation for ultrasensitive targeted SCP that requires both high sensitivity and robust quantitative performance.

DOI: https://doi.org/10.64898/2026.01.02.697431
Int J Mol Sci. 2025 Dec 23. pii: 149. [Epub ahead of print]27(1):

Mass Spectrometry Quantification of Epigenetic Changes: A Scoping Review for Cancer and Beyond.

Rossana Comito, Agnese Mannaioli, Agen Peter Lunghi Msemwa, Francesca Bravi, Carlotta Zunarelli, Eva Negri, Emanuele Porru, Francesco Saverio Violante.

  Mass spectrometry has become an indispensable tool for the identification and quantification of epigenetic modifications, offering both high sensitivity and structural specificity. The two major classes of epigenetic modifications identified-DNA methylation and histone post-translational modifications-play fundamental roles in cancer development, underscoring the relevance of their precise quantification for understanding tumorigenesis and potential therapeutic targeting. In this scoping review, we included 89 studies that met the inclusion criteria for detailed methodological assessment. Among these, we compared pre-treatment workflows, analytical platforms, and acquisition modes employed to characterize epigenetic modifications in human samples and model systems. Our synthesis highlights the predominance of bottom-up strategies combined with Orbitrap-based platforms and data-dependent acquisition for histone post-translational modifications, whereas triple quadrupole mass spectrometers were predominant for DNA methylation quantification. We critically evaluate current limitations, including heterogeneity in validation reporting, insufficient coverage of combinatorial post-translational modifications, and variability in derivatization efficiency.

Keywords:  DNA methylation; cancer biomarker; epigenetic modifications; histone post-translational modifications; mass spectrometry

DOI:  https://doi.org/10.3390/ijms27010149
STAR Protoc. 2026 Jan 03. pii: S2666-1667(25)00715-4. [Epub ahead of print]7(1): 104309

Protocol for stable isotopic tracing to assess cellular lipogenic activity in induced neural stem cells.

Rosana-Bristena Ionescu, Julie A Reisz, Monika Dzieciatkowska, Daniel Stephenson, Alexandra M Nicaise, Pranathi Prasad, Cory M Willis, Marta Suarez Cubero, Luca Peruzzotti-Jametti, Frank Edenhofer, Christian Frezza, Stefano Pluchino, Angelo D'Alessandro.

  Here, we present a protocol to assess the lipogenic phenotype of induced neural stem cells (iNSCs) using stable isotopic tracing. We describe steps for the culture and preparation of iNSCs, labeling with [13C6]-glucose and [13C5, 15N2]-glutamine, and the subsequent extraction of metabolites, lipids, and proteins from the same sample. This protocol supports single-specimen, mass spectrometry-based multi-omics workflows and is applicable to steady-state analyses, stable isotope tracing, and characterization of protein post-translational modifications. For complete details on the use and execution of this protocol, please refer to Ionescu et al.1.

Keywords:  metabolism; neuroscience; proteomics; protocols in metabolomics and lipidomics; stem cells

DOI:  https://doi.org/10.1016/j.xpro.2025.104309
Biomed Chromatogr. 2026 Feb;40(2): e70334

An Overview of Liquid Chromatography-Mass Spectrometry (LC-MS) Methods for the Quantification of Antibody-Drug Conjugates.

Pauline L M Buitelaar, Hilde Rosing, Jos H Beijnen, Neeltje Steeghs, Alwin D R Huitema.

  Antibody-drug conjugates (ADCs) are innovative drugs composed of cytotoxic molecules (payload) linked to antibodies, that selectively target and kill cancer cells upon internalization. In vivo, ADCs exist as intact molecules, naked antibodies, or released, unconjugated (linker-)payload. Accurate quantification of these entities is crucial for understanding ADCs pharmacokinetics. Ligand-binding assays are commonly used to measure ADC concentrations and total antibody concentrations, whereas LC-MS/MS is used to analyze the payload. Due to limitations in ligand-binding assays, this review focuses on quantitative LC-MS methods for the different ADC entities. Quantitative LC-MS assays were described for all ADC entities, available from full manuscripts and regulatory reviews of 12 ADCs evaluated by the European Medicine Agency, by January 2025. The review summarized sample pre-treatment, chromatography, mass spectrometry, validation, and stability data for each LC-MS method. Overall, critical details were often missing, particularly concerning sample pre-treatment, validation criteria, and sample stability. In conclusion, LC-MS quantification of ADC entities is feasible but current methods lack sufficient detail. Our review highlights the need for further research to develop reliable LC-MS assays for ADCs. This review may serve as a starting point and outlines key factors to consider in future LC-MS method development.

Keywords:  LC–MS; antibody‐drug conjugates; bioanalysis; literature review

DOI:  https://doi.org/10.1002/bmc.70334
Anal Bioanal Chem. 2026 Jan 06.

Sample preparation of Drosophila melanogaster larvae for MALDI imaging.

Marius Herbst, Marie A König, Katharina Gutbrod, Johannes Volk, Peter Dörmann, Margret H Bülow, Marianne Engeser.

  Mass spectrometry-based lipidomics has emerged as a crucial field for unraveling the complexity of biological systems through comprehensive profiling of lipid species with unparalleled sensitivity and specificity. Among the available techniques, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging, or MALDI-IMS) stands out as a powerful spatial lipidomics tool, enabling the visualization of distribution and localization of individual lipid classes and species directly within biological tissues. The spatial context of lipids provided by MALDI imaging is especially valuable for studying nutritional and developmental processes in heterogenic tissues. The reliability and quality of MALDI imaging data critically depend on sample preparation, which must preserve both tissue morphology and analyte integrity. Here we introduce a sample preparation protocol for Drosophila melanogaster larval tissue, addressing key limitations of standard protocols such as thin-sectioning. Owing to the small size of Drosophila tissues and the flat morphology of organs such as the fat body, our approach enables direct imaging of the inner organs of the larvae without sectioning. This not only streamlines the workflow and minimizes tissue disruption but also allows simultaneous analysis of all major internal organs in a single measurement. Our approach not only facilitates high-quality MALDI-IMS data acquisition from complex larval tissues but also enables the localization of a broad spectrum of lipid species complementary to data gained by established techniques like liquid chromatography mass spectrometry (LC-MS). These advances position MALDI imaging, when coupled with our optimized preparation workflow, as an indispensable technique for spatially resolved lipidomics studies in small model organisms such as Drosophila larvae.

Keywords:   Drosophila melanogaster ; MALDI imaging; Phospholipids; Triacylglycerides

DOI:  https://doi.org/10.1007/s00216-025-06289-x
Nature. 2026 Jan 07.

Nutrient requirements of organ-specific metastasis in breast cancer.

Keene L Abbott, Sonu Subudhi, Raphael Ferreira, Yetiş Gültekin, Sophie C Steinbuch, Muhammad Bin Munim, Diya L Ramesh, Sophie E Honeder, Ashwin S Kumar, Michelle Wu, Jacob A Hansen, Anna Shevzov-Zebrun, Edrees H Rashan, Kian M Eghbalian, Sharanya Sivanand, Anna M Barbeau, Lisa M Riedmayr, Mark Duquette, Ahmed Ali, Nicole Henning, Sonia E Trojan, Millenia Waite, Tenzin Kunchok, Mayu A Nakano, Florian Gourgue, Gino B Ferraro, Brian T Do, Virginia Spanoudaki, Francisco J Sánchez-Rivera, Xin Jin, George M Church, Rakesh K Jain, Matthew G Vander Heiden.

Cancer metastasis is a major contributor to patient morbidity and mortality1, yet the factors that determine the organs where cancers can metastasize are incompletely understood. Here we quantify the absolute levels of 124 metabolites in multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different tissue sites. We then asked how tumour growth in different tissues relates to nutrient availability and tumour biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. In addition, we identify purine synthesis as a requirement for tumour growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumour de novo nucleotide synthesis activity. These data suggest that a complex interplay between multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.

DOI: https://doi.org/10.1038/s41586-025-09898-9
Food Chem X. 2025 Dec;32 103360

Emerging analytical techniques for lipid profiling in food products: insights into processing effects and quality control.

Deepika Kathuria, Sonal Aggarwal, Akanksha Negi, Riya Barthwal, Aroma Joshi, Narpinder Singh.

  Lipids are essential components of foods which include fatty acids, glycolipids, sphingolipids, sterols, vitamin D, isoprenoids, etc. It possesses bioactive properties that exhibit desirable impact on human health. However, understanding the complexity of food lipids, requires sophisticated analytical methods capable of capturing their full spectrum. Lipidomics (LIP), an emerging omics field derived from metabolomics focuses on the complete analysis of lipid molecules within food matrices. Mass spectrometry (MS)-based LIP, exhibit great potential for analysing food lipids. The present review discusses the application of LIP in characterizing lipid metabolites in food, including how food processing, cooking, and storage, influence lipid profiles. Advancement in MS-based LIP enabled the detection of lipid alterations, along with ensuring food quality, authenticity, and safety. Future trends suggest further advancements in MS and separation techniques and application of Large Language Models will emerge as powerful tools in supporting oil processing research and industry practices. These techniques offered innovative predictions for mining significant information from lipidomic related large volumes of scientific and technical literature that will expand the application of LIP, providing deeper insights into lipid metabolism as well as understanding lipids in relation to health and nutrition.

Keywords:  Lipids; food; food processing; mass-spectrometry; metabolomic

DOI:  https://doi.org/10.1016/j.fochx.2025.103360
bioRxiv. 2025 Dec 27. pii: 2025.12.26.696642. [Epub ahead of print]

Improved surfactant-assisted one-pot sample preparation for robust convenient single cell proteomics.

Reta Birhanu Kitata, Zhangyang Xu, Nadia Bayou, Rui Zhao, William B Christler, Matthew J Gaffrey, Karl K Weitz, Mara Serena Serafini, Elisabetta Molteni, Vladislav A Petyuk, Massimo Cristofanilli, Tao Liu, Carolina Reduzzi, Tujin Shi.

Recent advances in mass spectrometry-based single-cell proteomics (SCP) technologies have revolutionized the SCP field for comprehensive characterization of cellular heterogeneity. However, most of the current SCP approaches employ sub-µL to 1 µL processing volume for effective single-cell sample preparation using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to the plate well to compensate water evaporation, which greatly limits their broad accessibility and analytical robustness. Here we report a robust convenient SCP method termed iSOP ( i mproved S urfactant-assisted O ne- P ot processing) for processing of single cells at the low µL processing volume using the 384-well plate with tight sealing to avoid sample drying loss. This iSOP SCP method was built upon our previously developed SOP method by the replacement of a PCR tube or 96-well plate with the low-volume 384-well plate and systematic optimization of the single-cell processing conditions. After optimization, 3 µL was selected as the processing volume with a mixture of 2 ng trypsin and 2 ng Lys-C enzymes in terms of robustness, detection sensitivity, and operation convenience. With a commonly accessible LC-MS platform, iSOP-MS can detect and quantify ∼1,200-1,800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines has demonstrated that iSOP-MS enabled reliable identification of an average of ∼1,700 and ∼2,050 protein groups from single BE2-C and SK-N-SH cells, respectively, and precise characterization of cellular heterogeneity between the two distinct cell types and within the same cell type. When compared to other available SCP methods, iSOP-MS is more robust and convenient for routine, cost-effective quantitative SCP analysis.

DOI: https://doi.org/10.64898/2025.12.26.696642
J Vis Exp. 2025 Dec 19.

Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2).

Giulia Barotti, Ronald Cutler, Joshua Cantlon, Barbara Montanini, Simone Sidoli.

Histone post-translational modifications (hPTMs) are central regulators of chromatin organization and gene expression. Their dysregulation is implicated in development, cancer, and aging. While mass spectrometry is the method of choice to study hPTMs, most protocols are designed for bulk samples and cannot resolve cell-to-cell variability. Extending histone PTM analysis to the single-cell level is therefore essential but technically challenging, as histones are low in abundance, lysine-rich, and heavily modified. Here, we describe an automated single-cell proteomics workflow for histone PTM analysis using nano liquid handling. The system enables nanoliter-scale processing of individual cells with minimal handling and high reproducibility. The workflow includes digestion with ArgC Ultra protease, which cleaves at arginine residues and avoids the need for lysine-blocking derivatization steps typically required in histone proteomics. To enable quantitative comparison between conditions, we apply a two-plex labeling strategy with propionic anhydride and its deuterated analogue, propionic anhydride-d10. This combination of automated sample preparation, isotopic multiplexing, and ArgC Ultra digestion results in a streamlined and sensitive protocol for single-cell histone PTM analysis. The method allows for the investigation of chromatin heterogeneity and the effects of epigenetic perturbations at single-cell resolution. To demonstrate the workflow, we generated spheroids from HepG2/C3A hepatocellular carcinoma cells and treated them with sodium butyrate, a histone deacetylase inhibitor that induces hyperacetylation.

DOI: https://doi.org/10.3791/69588
J Exp Clin Cancer Res. 2026 Jan 08.

BACE2 tunes lipid uptake through lipid transporters shedding supporting cancer cell proliferation.

Vittoria Matafora, Alice Elhagh, Alessandra Morelli, Laura Tronci, Angela Cattaneo, Camilla Conti, Francesca Casagrande, Francesco Farris, Angela Bachi.



Keywords:  BACE2; Cancer; Lipid droplets; Lipid uptake; Lipidomics; Proteomics

DOI:  https://doi.org/10.1186/s13046-025-03626-x
STAR Protoc. 2026 Jan 07. pii: S2666-1667(25)00731-2. [Epub ahead of print]7(1): 104325

Protocol for determining gut microbiota metabolites as substrates in mouse metabolism.

Han Fang, Dana Kukje Zada, Nicole G Barra, Rodrigo Rodrigues E-Lacerda, Jonathan D Schertzer.

  Here, we present a protocol for studying how microbiota-derived substrates fuel host mouse metabolism using stable isotope tracing. We describe steps for integrating 13C tracing in bacterial culture with 13C-labeled CO2 tracing in free-moving mice. We then detail procedures for determining the metabolic flux and fate of gut microbiota-derived substrates in the host through comparison between germ-free mice and monocolonized or specific pathogen-free mice. This protocol provides a framework for determining metabolic interactions between the gut microbiota and host. For complete details on the use and execution of this protocol, please refer to Fang et al.1.

Keywords:  mass spectrometry; metabolism; microbiology

DOI:  https://doi.org/10.1016/j.xpro.2025.104325
Anal Chem. 2026 Jan 09.

13C-Isotope Tracing Structural Lipidomics for Resolving Phospholipid Metabolism Dynamics in Human Breast Cancer Cells.

Zhuoning Xie, Qirui Yu, Simin Cheng, Dan Li, Zheng Ouyang, Xiaoxiao Ma.

Phospholipid metabolic homeostasis is critical, yet its dynamic regulation at the structural level remains poorly characterized, particularly from the perspective of metabolic flux analysis. This study presents an RPLC-PB-MS/MS workflow integrating 13C-glucose isotope tracing and the Paternò-Büchi (PB) reaction for comprehensive investigation of de novo phospholipid synthesis. By precisely mapping 13C labeling sites, we revealed distinct metabolic rates for various PL structural components. The glycerol backbone showed the fastest metabolism, while within phospholipid acyl chains, SFAs showed a greater contribution to 13C labeling compared to MUFAs. Furthermore, we quantified the labeling rates of PL C═C location isomers, highlighting dynamic regulation at the C═C location level. Notably, certain low-abundance n-7 isomers exhibited rapid turnover, suggesting potential functional roles in membrane remodeling or signaling. Analysis of three human breast cancer cell lines (MCF-7, MDA-MB-468, and BT-474) further revealed significant differences in PL metabolism dynamics, with MCF-7 cells showing initially rapid but transient labeling after passage, while MDA-MB-468 cells maintained sustained high fluxes. These findings provide insights into the structural specificity and dynamic regulation of phospholipid metabolism in cancer, offering potential therapeutic implications.

DOI: https://doi.org/10.1021/acs.analchem.5c06789
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Dec 31. pii: S1570-0232(25)00465-9. [Epub ahead of print]1270 124911

Quantitative GC-MS/MS in the clinical analysis of eicosanoids: A critical review and discussion, a 40-years historical retrospect, and possible implications for LC-MS/MS.

Dimitrios Tsikas.

  Mass spectrometry was a protagonist in the discovery of prostaglandins, thromboxane, leukotrienes and other arachidonic acid-derived molecules, collectively known as the eicosanoids. Mass spectrometry has played a significant role in exploration of their metabolic pathways in humans and animals, in health and disease, and in pharmacotherapy. Clinical researchers in the United States of America and in Europe, in close cooperation with chemist analysts, were the pioneers in the application of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) to quantitate eicosanoids and index metabolites in plasma, serum and urine samples from clinical trials by using stable-isotope labeled analogs as internal standards. In the present article, the application of the stable-isotope dilution GC-MS/MS methodology in the quantitative clinical analysis is reviewed. The focus is on prostaglandins, thromboxane, leukotrienes, and their so-called index metabolites for renal and whole-body synthesis of certain eicosanoids such as PGE2 and its major urinary metabolite (PGE-MUM), respectively. Nowadays, LC-MS/MS, which evolved later than GC-MS/MS, is increasingly used in numerous areas of research, including the eicosanoids in clinical studies. The present work critically discusses the current practice of LC-MS/MS users in the quantitative analysis of eicosanoids in biological samples. While the LC-MS/MS technology offers rapidity and high-throughput analysis, especially due to the renunciation of time-consuming analytical derivatization steps that are required in GC-MS/MS, LC-MS/MS seems to lack sufficient analytical sensitivity, i.e., lower limit of quantitation, for many eicosanoids such as thromboxane B2 and leukotriene B4. Reported data on basal concentrations of certain eicosanoids in plasma and urine samples from healthy humans as determined by LC-MS/MS are several orders of magnitude higher than originally reported by pioneering eicosanoid researchers, who developed, validated and used sophisticated, tailored GC-MS- and GC-MS/MS-based analytical methods for individual eicosanoids. Modern eicosanoids researchers would greatly benefit from the milestones and signposts set previously eicosanoids researchers from the very start. A key milestone and signpost is the concentration of primary eicosanoids and their metabolites in plasma, serum and urine samples of healthy humans. Issues for consideration in the GC-MS/MS and LC-MS/MS analysis of eicosanoids are discussed.

Keywords:  Eicosanoids; Index metabolites; Isoprostanes; Leukotrienes; Mass spectrometry; Prostaglandins; Quantitation; Thromboxane

DOI:  https://doi.org/10.1016/j.jchromb.2025.124911