bims-mascan 2026-08-09 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2026–08–09
34 papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Generation of High-Quality Succinyl Spectral Libraries for Improved Proteome-Wide Succinylome Analysis Using Data-Independent Acquisition.
Evaluation of a Targeted LC-MS/MS Assay for Clinical Quantification of Urinary Organic Acids.
Mass Spectrometry-Based Extracellular Vesicle Proteomics for Biomarker Discovery.
Comparative Evaluation of Adaptive Focused Acoustics- and SP3-Based Workflows for Deep Proteomic Profiling of 30-Year-Old Formalin-Fixed Paraffin-Embedded Tissues.
O- and N-Phosphoproteomic Analysis Using Online Alkaline-pH Reversed-Phase Nanoelectrospray-Tandem Mass Spectrometry.
Deep intact proteoform quantification of low-microgram-scale proteome by tandem mass tag labeling and online 2D high-pH/low-pH RPLC top-down analysis.
Pyridyl ethyl diazoacetate derivatization enables LC-MS annotation of cholesterol and its metabolites.
Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis.
Systematic evaluation of PASEF acquisition strategies in complex metaproteomes.
Systematic Optimization of an SLE-DAPTAD LC-MS/MS Workflow for Simultaneous Quantification of Low-Abundance Vitamin D3 Metabolites in Plasma.
PEPPI-SP3: A Gel-Based High-Resolution Sample Preparation Workflow for In-Depth Top-Down Analysis of Intact Proteoforms by Mass Spectrometry.
Phosphoproteomics Protocol for the Identification of Novel Biomarkers from Extracellular Vesicles of Cancerous Cells.
Decoding the SUMO Proteome: A Mass Spectrometry Workflow for Site-Specific Identification.
HILIC-MS/MS-based strategy for quantitative amino acid profiling in multiple biological matrices and its application in kidney disease samples.
Extending singlet oxygen lifetime to enhance sensitivity of isomer-selective lipid imaging using nanospray desorption electrospray ionization (nano-DESI).
Development and validation of an LC-MS method based on derivatization for quantification of kynurenine pathway metabolites in serum and application in cancer patients.
Uncovering PFAS and halogenated chemicals using nontargeted analysis and the PFAS IMplementer for mass spectrometry (PIMMS).
Optimization and Validation of Plasma-Based Assay for Detection and Quantification of Monoamine Neurotransmitter Metabolites Using LC-MS/MS.
An Overview of Mass Spectrometry-Based Methods to Analyze Post-Translational Modifications.
Mannose supports lung cancer metabolism during glycolytic limitation.
Enrichment and Metaproteomic Analysis of Lysine Acetylation in Fecal Microbiome Samples.
Palmitate promotes liver metastases by decreasing neutrophil antitumour behaviour.
Beyond bar charts: A practical guide to small sample pharmacological data.
Fit-for-purpose analytics for cannabis: identification, quantification, and standardization of cannabinoids, flavonoids and terpenes.
Quantifying the aromatic amino acid metabolome: UPLC-MS/MS analysis of aromatic amino acids and their host and co-metabolites in plasma.
Mapping Cellular Protein Lysine Methylation Using Targeted-Mass Spectrometry.
Profiling O-Acetylation in Sialoglycans Using MALDI-TOF and LC-MS/MS with Methylamidation and Permethylation Derivatization.
Development and validation of a sensitive HPLC-MS/MS method for the analysis of semaglutide in human plasma.
Combining SDS-PAGE with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics Analysis of Intact Histone Proteoforms.
Remodeling of lipid metabolism orchestrates human adipose-derived mesenchymal stem cells osteodifferentiation.
An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of meropenem in human serum and plasma.
A targeted lipidomics workflow for high-throughput phenotyping and mechanism-based in vitro hepatotoxicity studies.
NNMT Loss Drives Cancer Progression by enhancing SAM availability for mTORC1 Signaling and Chromatin Methylation.
Computational metabolomics at scale: from open data to insight.

Methods Mol Biol. 2026 ;3018 169-181

Generation of High-Quality Succinyl Spectral Libraries for Improved Proteome-Wide Succinylome Analysis Using Data-Independent Acquisition.

Alice Zhang, Birgit Schilling, Joanna Bons.

  Protein posttranslational modifications (PTMs) are crucial and dynamic modulators of protein functions, interactions, and localizations, as well as biological pathways and cellular signaling. Lysine succinylation analysis remains challenging, but sophisticated workflows combining succinylated peptide enrichments and quantitative mass spectrometry approaches have revolutionized proteome-wide succinylome analysis. The implementation of data-independent acquisition (DIA)-mass spectrometry has greatly advanced the detection of low-abundance succinylated peptides, succinylome coverage, identification reproducibility, and quantification accuracy. However, the complexity of DIA data requires dedicated data processing algorithms and tools, which typically rely on spectral libraries. These reference libraries can be generated from experimental data-dependent acquisition (DDA) acquisitions of representative study samples submitted to DDA database search engines for confident succinylated peptide identification and precise PTM site localization. Here, we describe how to build DDA PTM spectral libraries using various software tools, specifically Spectronaut, SpectroMine, and MSFragger. The generated libraries were imported into Skyline for the analysis of previously published DIA succinylome data of Sirtuin-5 knocked-out vs wild-type mouse brains in order to accurately quantify and visualize PTM-containing peptides.

Keywords:  Data-dependent acquisition; Data-independent acquisition; Posttranslational modifications; Quantitative proteomics; Spectral library; Succinylation

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_11
J Mass Spectrom. 2026 Aug;61(8): e70099

Evaluation of a Targeted LC-MS/MS Assay for Clinical Quantification of Urinary Organic Acids.

Earnest J P Daniel, Emily Garnett, Deepthi Rajapakshe, Sridevi Devaraj.

Gas chromatography-mass spectrometry (GC-MS) is the reference method for urine organic acid analysis but requires complex sample preparation and derivatization, limiting routine clinical use. We developed and validated a targeted Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying urinary organic acids relevant to the evaluation of inborn errors of metabolism. The assay demonstrated excellent linearity, precision, accuracy, analytical sensitivity, and good agreement with GC-MS. Organic acidurias and other metabolic disorders were accurately identified through characteristic metabolite elevations. The simplified workflow eliminates derivatization and enables rapid, high-throughput implementation for newborn screening confirmation, metabolic screening, and patient monitoring in pediatric clinical laboratories.

DOI: https://doi.org/10.1002/jms.70099
Mass Spectrom Rev. 2026 Aug 03.

Mass Spectrometry-Based Extracellular Vesicle Proteomics for Biomarker Discovery.

Ya-Juan Liu, Juan Peng, Chao Kang.

  Extracellular vesicle (EV) proteomics has emerged as a powerful platform for decoding intercellular communication and advancing biomarker discovery across human diseases. EVs carry proteins that reflect their cells of origin, offering a minimally invasive window into physiological and pathological processes. Mass spectrometry (MS) now enables deep, high-resolution EV proteome profiling, aided by improved isolation and rigorous characterization that ensure sample purity and integrity. Advanced computational pipelines integrating quantitative modeling, spectral-library prediction, machine learning and multi-omics analysis extract meaningful biological signals, revealing subtle disease-associated EV signatures and establishing EV proteomics as a strong platform for biomarker discovery and precision medicine. This review provides an integrated framework linking EV isolation principles, characterization strategies, mass-spectrometric workflows, and computational analysis to the biological and clinical insights they generate. We also highlight key challenges and future directions, including the need for standardized reference materials, unified pre-analytical workflows, and EV proteome reference atlases. Together, these innovations are transforming EV proteomics into a next-generation tool for precision medicine.

Keywords:  biomarker; extracellular vesicle; mass spectrometry; proteomics

DOI:  https://doi.org/10.1002/mas.70040
Proteomics. 2026 Aug 06. e70169

Comparative Evaluation of Adaptive Focused Acoustics- and SP3-Based Workflows for Deep Proteomic Profiling of 30-Year-Old Formalin-Fixed Paraffin-Embedded Tissues.

Rei Noguchi, Masatsugu Ishii, Yuki Adachi, Yuki Yoshimatsu, Kazuki Sasaki, Nobuyoshi Hiraoka, Sumio Ohtsuki, Tadashi Kondo.

  Formalin-fixed paraffin-embedded (FFPE) tissues represent a vast archive for translational research that offers access to specimens with clinical outcomes. However, formalin-induced crosslinking hinders efficient protein extraction and limits proteomic applications. We systematically compared three protein extraction workflows, adaptive focused acoustics (AFA) with Covaris buffer (AFA/Covaris-b), AFA with SDS buffer (AFA/SDS-b), and SDS-based lysis followed by SP3 clean-up and digestion (SP3/SDS-b), using hepatocellular carcinoma (HCC) tissues archived for over 30 years. Benchmarking was performed on two HCC cases using three serial tumor sections each, followed by protein quantification using data-independent acquisition mass spectrometry on a ZenoTOF 7600+ instrument. All workflows identified 5206 proteins, with similar mean identifications (AFA/Covaris-b: 4068; AFA/SDS-b: 4134; SP3/SDS-b: 4048), but distinct reproducibility (CVs: 11.9%, 12.2%, and 20.8%, respectively), leading to the exclusion of SP3. Method selection across eight HCC cases, including paired tumor and adjacent non-tumorous tissues, revealed a total of 6018 proteins, with mean identifications of 3876 (AFA/Covaris-b) and 3852 (AFA/SDS-b). Both AFA workflows showed high reproducibility, with slightly lower variability for AFA/Covaris-b (CVs: HCC: 13.5%, non-tumorous liver tissue (NTL): 11.0%) compared with AFA/SDS-b (CVs: HCC: 14.8%, NTL: 11.3%). While upregulated pathways were not consistently detected across workflows, downregulated pathways showed greater concordance between methods. These findings should be interpreted cautiously given the lack of appropriate temporal or fresh-frozen controls. Collectively, AFA-based workflows enable reproducible proteomic profiling of long-archived FFPE tissues and provide a practical approach for methodological evaluation in retrospective proteomics studies. SIGNIFICANCE OF THE STUDY: FFPE tissues are one of the most valuable clinically annotated biospecimen resources worldwide. However, their routine use in proteomics is hindered by extensive protein cross-linking, particularly in long-term archived blocks. Our study addresses this challenge by systematically evaluating and validating protein extraction strategies for FFPE hepatocellular carcinoma tissues stored for more than 30 years. By directly benchmarking AFA-based and SP3 workflows, we show that AFA protocols, particularly those with the Covaris buffer, provide superior reproducibility while maintaining broad proteome coverage. Importantly, we showed that archival samples yielded more than 6,000 quantifiable proteins, with reproducible pathway-level patterns observed across methods, although interpretation of biological signals remains limited by the absence of appropriate controls and potential extraction-related biases. These findings suggest that long-preserved FFPE specimens can serve as useful resources for modern data-independent acquisition mass spectrometry. The ability to extract reproducible proteomic information from decades-old FFPE blocks has important implications; it enables retrospective biomarker discovery, integration with genomic data, and validation of therapeutic targets in richly annotated patient cohorts. By providing a reproducible workflow, this study supports the expanded use of archived FFPE tissues and facilitates future efforts for large-scale, longitudinal cancer proteomics.

Keywords:  SP3; adaptive focused acoustics; hepatocellular carcinoma; long archived formalin‐fixed paraffin‐embedded; proteomics

DOI:  https://doi.org/10.1002/pmic.70169
Methods Mol Biol. 2026 ;3018 111-122

O- and N-Phosphoproteomic Analysis Using Online Alkaline-pH Reversed-Phase Nanoelectrospray-Tandem Mass Spectrometry.

Yuqiu Wang, Minchu Tang, Wenfan Xie.

  Protein phosphorylation (O-linked and N-linked) is associated with a wide range of biological processes and cell signaling pathways. Aberrant phosphorylation is often observed as a hallmark of diseases, including cancer, neurodegenerative disorders, and metabolic syndromes. Mass spectrometry (MS) is an indispensable tool for studying protein phosphorylation. This is primarily due to its high sensitivity and throughput, which allows for comprehensive identification and quantification of phosphorylation events in complex biological samples. In this chapter, we describe a detailed protocol for phosphoproteomic analysis using one-dimensional online alkaline-pH reversed-phase nanoelectrospray-tandem MS (alkaline-pH-MS/MS). It complements traditional online low-pH reversed-phase nanoelectrospray-tandem MS (low-pH-MS/MS) by modulating the charge state distribution of phosphopeptides, which may facilitate the characterization of phosphoproteins with significant biological functions.

Keywords:  Alkaline-pH-MS/MS; Mass spectrometry; Phosphoproteomics; Phosphorylation; Posttranslational modification

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_7
Anal Chim Acta. 2026 Oct 08. pii: S0003-2670(26)00796-8. [Epub ahead of print]1418 345846

Deep intact proteoform quantification of low-microgram-scale proteome by tandem mass tag labeling and online 2D high-pH/low-pH RPLC top-down analysis.

Yanting Guo, Trishika Chowdhury, Walter P Galie, Kellye A Cupp-Sutton, Zhitao Zhao, Ashlyn Solenberger, Dahang Yu, Si Wu.

  Isobaric chemical tag labels are the 'gold standard' for quantifying proteins in bottom-up proteomics. Recently, our group developed and optimized an intact protein-level tandem mass tag (TMT) labeling platform to identify and quantify intact proteoforms in complex biological samples. This intact-protein TMT labeling strategy enables multiplexed quantification and minimizes variability introduced during downstream sample preparation. Here, to achieve deeper proteome coverage, we developed an integrated TMT-labeling and online 2D high-pH/low-pH RPLC top-down workflow for proteoform quantification in complex cell lysates. TMT-labeled intact proteoforms derived from HeLa lysate were mixed at defined ratios and analyzed using the developed platform. The measured TMT reporter ion ratios for detected intact proteoforms closely matched the expected theoretical values, demonstrating high quantitative accuracy. The platform was further applied to quantify changes in proteoform abundance induced by staurosporine (STS) in HeLa cells. Using six channels of TMT10plex reagents, with 3.3 μg of protein labeled per channel and 20 μg total protein injected, we identified 47 proteoforms from 21 proteins exhibiting significant abundance changes in microgram-level samples. Functional enrichment analysis using DAVID associated these proteoforms with metabolic and apoptotic pathways previously linked to STS-induced cellular responses. These results underscore the platform's ability to resolve biologically relevant proteoform-level regulation. This work establishes a multiplexed, quantitative, multidimensional top-down proteomics platform that enables deep proteoform characterization using only microgram-level sample amounts. Data are available via ProteomeXchange with identifier PXD078120.

Keywords:  Isobaric labeling; Online 2D-LC; Protein-level TMT labeling; Top-down proteomics

DOI:  https://doi.org/10.1016/j.aca.2026.345846
Anal Chim Acta. 2026 Oct 08. pii: S0003-2670(26)00805-6. [Epub ahead of print]1418 345855

Pyridyl ethyl diazoacetate derivatization enables LC-MS annotation of cholesterol and its metabolites.

Wenhao Ke, Yikang Wang, Yang Chen, Hanchu Ye, Wen-Di Duan, Feifei Lin, Yingying Wang, Zhengjun Zhou, Zhiyong Yu, Youhong Hu, Jia Liu.

  Cholesterol and its metabolites are fundamental to membrane organization and cellular signaling, but their poor ionization efficiency and structural similarity complicate mass-spectrometric (MS) analysis. In this work, we developed pyridyl ethyl diazoacetate (PED) as a stable, efficient derivatization reagent that introduces a permanent positive charge on hydroxyl-containing sterols, thereby enhancing ionization and detection sensitivity. Systematic MS2 characterization established diagnostic fragmentation motifs of PED-tagged sterols, which we encoded into a query-based spectral-mining strategy for reproducible, rule-based annotation of sterol features in complex matrices. Application of the PED workflow to human liver tissues enabled sterol profiling across cirrhotic, hepatocellular carcinoma, and intrahepatic cholangiocarcinoma specimens, suggesting disease-context-dependent differences in sterol profiles. The workflow effectively integrates chemical derivatization and programmable data interrogation, providing a robust analytical framework for sterol profiling in biological samples and a potential basis for broader lipidomic applications.

Keywords:  Cholesterol derivatization; Liver disease metabolomics; Mass spectrometry; Pyridyl ethyl diazoacetate; Spectral mining; Sterol profiling

DOI:  https://doi.org/10.1016/j.aca.2026.345855
Elife. 2026 Aug 05. pii: RP106492. [Epub ahead of print]14

Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis.

Patrick B Jonker, Mumina Sadullozoda, Guillaume Cognet, Juan J Apiz Saab, Kelly H Sokol, Violet X Wu, Deepa Kumari, Colin Sheehan, Mete E Ozgurses, Darby Agovino, Grace Croley, Lindsey N Dzierozynski, Chufan Cai, Leah M Ziolkowski, Smit A Patel, Althea Bock-Hughes, Kay F Macleod, Hardik Shah, Jonathan L Coloff, Evan C Lien, Alexander Muir.

  Nutrient limitation is a characteristic feature of poorly perfused tumors. In contrast to well-perfused tissues, nutrient deficits in tumors impose metabolic constraints on cancer cells. The metabolic constraints created by the tumor microenvironment can lead to vulnerabilities in cancers. Identifying the metabolic constraints of the tumor microenvironment and the vulnerabilities that arise in cancers can provide new insight into tumor biology and identify promising anti-neoplastic targets. To identify how the microenvironment constrains the metabolism of pancreatic tumors, we challenged pancreatic cancer cells with microenvironmental nutrient levels and analyzed changes in cellular metabolism. We found that arginine limitation in pancreatic tumors perturbs saturated and monounsaturated fatty acid synthesis by suppressing the lipogenic transcription factor SREBP1, in part via activation of the amino acid sensor GCN2. Synthesis of these fatty acids is critical for maintaining a balance of saturated, monounsaturated, and polyunsaturated fatty acids (PUFAs) in cellular membranes. Because of microenvironmental constraints on fatty acid synthesis, pancreatic cancer cells and tumors are unable to maintain lipid homeostasis when exposed to PUFAs, leading to cell death by ferroptosis. In sum, arginine restriction in the tumor microenvironment constrains lipid metabolism in pancreatic cancers, which renders these tumors vulnerable to polyunsaturated-enriched fats.

Keywords:  biochemistry; cancer; cancer biology; chemical biology; diet; human; metabolism; mouse; stress; synthetic lethality; tumor microenvironment

DOI:  https://doi.org/10.7554/eLife.106492
Nat Commun. 2026 Aug 03. pii: 7406. [Epub ahead of print]17(1):

Systematic evaluation of PASEF acquisition strategies in complex metaproteomes.

Feng Xian, Goran Mitulovic, Ranjith Kumar Ravi Kumar, Lukas Uhrik, Elisabeth Urbauer, Doriane Aguanno, Dirk Haller, Manuela Schmidt, David Gomez-Varela.

Metaproteomics measures functional expression in complex microbial communities, but extreme sample complexity and dynamic range challenge acquisition strategies. Trapped ion mobility spectrometry with parallel accumulation-serial fragmentation (PASEF) has expanded into multiple acquisition modes, yet systematic evaluations in high-complexity metaproteomes remain limited. Here, we benchmark five PASEF modes-DDA-, DIA-, Slice-, Synchro-, and midia-PASEF-using a complex fecal peptide background spiked with defined bacterial references. Across three gradients and input levels, 540 LC-MS acquisitions are analyzed under matched conditions. Based on data-derived performance scores, DIA-based strategies outperform DDA-PASEF in peptide and protein coverage, particularly for low-abundance microbial features. DIA- and Slice-PASEF show strong quantitative reproducibility, reduced ratio compression, and consistent species-abundance scaling, while functional profiling reveals expanded annotation depth. When tested in a murine colonic injury model, the two highest-scoring methods, DIA- and Slice-PASEF, capture concordant host and microbial responses.

DOI: https://doi.org/10.1038/s41467-026-75845-5
Rapid Commun Mass Spectrom. 2026 Oct 30. 40(20): e70158

Systematic Optimization of an SLE-DAPTAD LC-MS/MS Workflow for Simultaneous Quantification of Low-Abundance Vitamin D3 Metabolites in Plasma.

Marzieh Amirmostofian, Michael D Wiese, Paul H Anderson, Peter Hoffmann.

   RATIONALE: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vitamin D3 metabolites is analytically challenging because these compounds are structurally similar, strongly matrix-associated, and have poor ionization efficiency. These limitations are particularly important for low-abundance metabolites such as 1,25(OH)2D3. This study systematically compared extraction and derivatization strategies to develop a simplified workflow for simultaneous quantification of 25(OH)D3, 3-epi-25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3 in plasma.
METHODS: Five sample-preparation approaches-simple protein precipitation, liquid-liquid extraction, salting-out assisted liquid-liquid extraction, solid-phase extraction, and supported liquid extraction-were evaluated under comparable conditions. Two derivatization reagents, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS), were compared. The selected workflow was validated according to bioanalytical method validation criteria.
RESULTS: Supported liquid extraction using ethyl acetate/methyl tert-butyl ether (1:1, v/v) provided the best overall balance of signal intensity, reproducibility, simplicity, and suitability for high-throughput processing. DAPTAD derivatization produced a substantially higher MS response than FMP-TS and was selected for the final method. The optimized SLE-DAPTAD LC-MS/MS workflow showed good linearity (r2 > 0.99), acceptable accuracy and precision, recovery of 72.5%-104%, and limits of quantification of 0.08 ng/mL for 3-epi-25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3, and 0.88 ng/mL for 25(OH)D3.
CONCLUSIONS: The optimized SLE-DAPTAD LC-MS/MS workflow provides a simple, sensitive, and automation-compatible approach for simultaneous quantification of four vitamin D3 metabolites in plasma. The method is positioned as an analytical workflow for method development and low-volume plasma analysis, rather than as a fully standardized routine diagnostic assay for endogenous 1,25(OH)2D3 measurement.

Keywords:  DAPTAD derivatization; LC–MS/MS; method optimization; sample preparation; supported liquid extraction; vitamin D3 metabolites

DOI:  https://doi.org/10.1002/rcm.70158
Methods Mol Biol. 2026 ;3018 199-209

PEPPI-SP3: A Gel-Based High-Resolution Sample Preparation Workflow for In-Depth Top-Down Analysis of Intact Proteoforms by Mass Spectrometry.

Ayako Takemori, Jake T Kline, Luca Fornelli, Nobuaki Takemori.

  Top-down proteomics enables large-scale identification of proteoforms by analyzing intact proteins extracted from biological samples using mass spectrometry systems coupled with liquid chromatography or capillary electrophoresis. In this chapter, we present the PEPPI-SP3 workflow, a high-resolution sample prefractionation method that significantly enhances the analytical depth of top-down proteomics by combining highly efficient passive extraction of in-gel proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with SP3 bead-based purification of intact proteins.

Keywords:  PEPPI-MS; Proteoforms; SDS-PAGE; SP3; Sample prefractionation; Top-down proteomics

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_13
Methods Mol Biol. 2026 ;3018 123-137

Phosphoproteomics Protocol for the Identification of Novel Biomarkers from Extracellular Vesicles of Cancerous Cells.

Zoran Minic, Suttinee Poolsup, Yingxi Li, Rochelle D'Mello, Maxim V Berezovski.

  In this chapter, we describe a general sample preparation workflow for small extracellular vesicles (sEVs) from breast cancer cell lines using techniques such as protein solubilization, enzymatic digestion, and Fe3+-immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) enrichment prior to mass spectrometry-based proteomic analysis. This workflow can be used to perform global phosphoproteomics of sEVs enabling the study of tumor biology and the discovery of biomarkers for cancer diagnosis and treatment.

Keywords:  Cells and sEVs preparation; IMAC phosphopeptide enrichment; Protein extraction; Titanium dioxide phosphopeptide enrichment; Tryptic digestion

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_8
Methods Mol Biol. 2026 ;3018 41-56

Decoding the SUMO Proteome: A Mass Spectrometry Workflow for Site-Specific Identification.

Chongyang Li, Pierre Thibault.

  Protein SUMOylation is a dynamic post-translational modification that regulates numerous cellular processes, including DNA repair, transcription, and proteostasis. SUMO modifiers are conjugated to lysine residues on substrate proteins via a conserved enzymatic cascade and can form diverse chain architectures that encode specific cellular outcomes. The identification of SUMOylated proteins and their modification sites has historically been challenging due to the low abundance of SUMOylation and the complexity of SUMO remnants after proteolysis. Recent advances in proteomics have led to the development of enrichment strategies and mass spectrometry (MS)-based methods that now enable the site-specific mapping of SUMO modifications. This chapter provides an overview of the biological roles and structural diversity of SUMOylation, and presents an MS-based workflow designed to identify SUMOylation sites with high specificity and depth. These tools offer new opportunities to dissect the SUMO-modified proteome in health and disease.

Keywords:  Immunoaffinity enrichment; Mass spectrometry; Posttranslational modifications; Proteomics; SUMOylation; Ubiquitin-like proteins (UBLs)

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_3
Anal Bioanal Chem. 2026 Aug 04.

HILIC-MS/MS-based strategy for quantitative amino acid profiling in multiple biological matrices and its application in kidney disease samples.

Xiongwei Yin, Jean-Paul Decuypere, Djalila Mekahli, Bert Bammens, Rudi Vennekens, Erwin Adams, Ann Van Schepdael.

  Amino acid metabolism disorders have strong links to various kidney diseases, and the kidney is essential for maintaining systemic amino acid homeostasis. Given the lack of obvious clinical symptoms in the early stages of kidney disease, searching for reliable metabolic biomarkers is essential for early diagnosis. Endogenous AAs are widely present in human biological matrices, making it difficult to obtain analyte-free blank matrices for method validation. This poses challenges in the preparation of calibration standards, leading to uncertainty and reduced consistency in the results. This study aimed to develop a rapid hydrophilic interaction liquid chromatographic method coupled with tandem mass spectrometry. It could analyze 47 amino acids and related compounds, present in various biological matrices (human plasma, urine, and cyst fluids). The calibration curves were constructed by stripping each matrix to obtain a surrogate matrix. The slopes of the different calibration curves were systematically evaluated and the analytical results were compared with results obtained through the method of standard addition. The calibration curves established using both the standard addition method and the matrix stripping method were parallel, hence matrix stripping effectively mitigates matrix effects under a variety of matrix conditions, thereby assuring the accuracy and reliability of quantitative analysis results. To further investigate the applicability of this method to large-scale sample analysis, the analysis of plasma samples and urine samples obtained from patients with two types of kidney disease was performed.

Keywords:  Amino acids; Cyst fluid; LC-MS/MS; Matrix effect; Plasma; Urine

DOI:  https://doi.org/10.1007/s00216-026-06687-9
Anal Chim Acta. 2026 Oct 08. pii: S0003-2670(26)00817-2. [Epub ahead of print]1418 345867

Extending singlet oxygen lifetime to enhance sensitivity of isomer-selective lipid imaging using nanospray desorption electrospray ionization (nano-DESI).

Sara Amer, Daisy Unsihuay, Emerson Hernly, Alessandra Latorre, Julia Laskin.

  Identifying and localizing lipid CC positional isomers in biological tissues remains a major analytical challenge for isomer-selective mass spectrometry imaging (iMSI). Herein, we report a straightforward yet powerful strategy to enhance the efficiency of singlet oxygen (1O2) reactions used for iMSI of lipids by extending the 1O2 lifetime using deuterated solvents. Replacing the conventional 9:1 (v/v) MeOH/H2O nanospray desorption electrospray ionization (nano-DESI) solvent with its deuterated counterpart, CD3OD/D2O, enhances the efficiency of lipid hydroperoxide (LOOH) formation by at least a factor of two. The resulting increase in signals of diagnostic fragments of LOOHs enhances sensitivity of nano-DESI iMSI for fatty acids and both mono- and polyunsaturated lipid species. Using this optimized solvent system, we achieved a deeper molecular coverage of isomeric lipids in nano-DESI iMSI of mouse brain tissue, enabling the detection and spatial mapping of lipid isomers previously undetectable with MeOH/H2O. Overall, the use of deuterated solvents is a practical and broadly applicable strategy for enhancing the sensitivity and coverage of photochemical derivatization-based nano-DESI iMSI, which will offer new insights into lipid structure and spatial distribution in complex biological systems.

Keywords:  Double-bond localization; Lipid hydroperoxide; Lipid isomers; Mass spectrometry imaging; Photochemical derivatization; Singlet oxygen

DOI:  https://doi.org/10.1016/j.aca.2026.345867
J Chromatogr A. 2026 Jul 26. pii: S0021-9673(26)00629-1. [Epub ahead of print]1785 467301

Development and validation of an LC-MS method based on derivatization for quantification of kynurenine pathway metabolites in serum and application in cancer patients.

Kinga Ożga-Wybranowska, Paweł Stepuch, Inga Łopuszyńska, Marek Durlik, Elżbieta Radzikowska-Büchner, Ryszard Maciejewski, Ilona Sadok.

  Simultaneous determination of a broad panel of kynurenine pathway metabolites remains analytically challenging due to their diverse polarity, poor chromatographic retention, and wide concentration range in biological matrices. In this work, a liquid chromatography-mass spectrometry method, which includes a derivatization step to form ester derivatives of tryptophan metabolites, was developed to measure serum levels of kynurenine, kynurenic acid, 3-hydroxykynurenine, picolinic acid, nicotinic acid, quinolinic acid, and xanthurenic acid. Sample preparation procedure was thoroughly optimized. As part of method development, different strategies for sample purification were tested. Ultimately, serum samples after derivatization were subjected to clean-up using solid-phase extraction. This approach enables the separation of isomeric pairs (picolinic and nicotinic acids) and improved the detection of quinolinic acid. Data were normalized to a single internal standard (3-nitro-l-tyrosine), which undergoes derivatization in the same manner as the target analytes. The method demonstrated excellent linearity (R² > 0.99) over wide concentration ranges, typically from low nanomolar to several tens of micromolar levels. Low limits of quantification were determined in a surrogate matrix: 0.35 µM for picolinic acid, 0.11 µM for 3-hydroxykynurenine, and <80 nM for the other metabolites.Inter- and intraday accuracy ranged between 90-110%, with recoveries >70% for all analytes. The method was validated and successfully applied to the quantification of kynurenine pathway metabolites in serum from healthy donors and patients with gastric or pancreatic cancer.

Keywords:  3-hydroxykynurenine; Derivatization; Gastric cancer; Kynurenine; Pancreatic cancer; Picolinic acid; Quinolinic acid

DOI:  https://doi.org/10.1016/j.chroma.2026.467301
Sci Adv. 2026 Aug 07. 12(32): eaeg0781

Uncovering PFAS and halogenated chemicals using nontargeted analysis and the PFAS IMplementer for mass spectrometry (PIMMS).

Gregory P Kudzin, James N Dodds, Erin S Baker.

Per- and polyfluoroalkyl substances (PFASs) are synthetic chemicals of considerable epidemiological concern. While targeted liquid chromatography (LC)-tandem mass spectrometry (MS/MS) methods are established for legacy PFAS, emerging replacement compounds require nontargeted analysis (NTA) for comprehensive characterization. However, NTA of complex biological matrices like human serum is analytically challenging because of the presence of many endogenous interferences and unknown PFAS molecules. To address this, ion mobility spectrometry has been coupled with LC and MS (LC-IMS-MS). This approach provides orthogonal separation via collision cross section to resolve isobars and filter noise. Despite its utility, current software lacks efficient PFAS feature prioritization, requiring extensive manual curation. We developed the PFAS IMplementer for Mass Spectrometry (PIMMS), a vendor-neutral, open-source tool that uses advanced filtering and scoring algorithms for rapid LC-IMS-MS data analysis. The potential utility of PIMMS was demonstrated using serum from bovine and human populations. PIMMS could identify both legacy and novel PFAS in addition to eight halogenoalkane xenobiotics (containing bromine, chlorine, and iodine). Of note, these halogenoalkane species were often only singly or doubly halogenated and were still effectively prioritized by PIMMS demonstrating the potential to identify a potentially growing number of novel compounds with only a single degree of halogenation.

DOI: https://doi.org/10.1126/sciadv.aeg0781
ACS Omega. 2026 Jul 28. 11(29): 43625-43637

Optimization and Validation of Plasma-Based Assay for Detection and Quantification of Monoamine Neurotransmitter Metabolites Using LC-MS/MS.

Lloyd Tauro, Kusuma C G, Nirpendra Singh, Vykuntaraju K Gowda, Rakesh K Mishra, Runa Hamid.

One of the essential subclasses of biogenic amines is monoamine neurotransmitters, which include dopamine, serotonin, norepinephrine, and epinephrine neurotransmitters. The biosynthesis pathways of these neurotransmitters share several common substrates, metabolites, cofactors, and enzymes. Genetic mutations in these components lead to a group of rare inherited conditions termed monoamine neurotransmitter metabolic disorders (mNMDs). Existing studies report methods for measurement of a particular set of metabolites belonging to this pathway for diagnosis, frequently across different biological matrices such as cerebrospinal fluid (CSF), plasma, serum, or urine. Hence, the available assays and data are methodologically heterogeneous. In a biochemical pathway, disorder-specific metabolic patterns arise from paired alterations in multiple upstream and downstream metabolites, and these patterns can be captured only when these metabolites are measured simultaneously from the same biological matrix under similar analytical conditions. The lack of concurrent measurement limits the use of metabolite ratios, which can be of diagnostic value and pattern-based interpretation in the case of mNMDs. In this study, we report the development, optimization, and validation of a plasma-based targeted liquid chromatography-mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 11 key metabolites of the monoamine biosynthesis pathway. The assay was optimized for sample preparation, chromatographic separation, MS detection, and validated according to the M10 Bioanalytical Method Validation and Study Sample Analysis, US -FDA guidelines (November 2022). The method demonstrates good linearity across a broad range, high sensitivity and specificity, acceptable interday- and intraday-precision and accuracy, acceptable coefficient of variation, and lower limit of detection and quantification. The applicability of the method was further validated using plasma samples from healthy participants. As mNMDs are potentially treatable if identified early, this validated plasma-based method can provide a quick and predictive alternative to currently used different biological matrices or invasive CSF sampling for diagnosis with additional utility for longitudinal therapeutic monitoring of these treatable conditions.

DOI: https://doi.org/10.1021/acsomega.6c02312
Methods Mol Biol. 2026 ;3018 3-21

An Overview of Mass Spectrometry-Based Methods to Analyze Post-Translational Modifications.

Rui Chen, Xu Zhang, Jianjun Li.

  Post-translational modifications (PTM) are the covalent modifications of proteins after their biosynthesis. PTMs change the structure and functions of proteins, which eventually regulate various biological processes that are closely related to the onset and development of diseases. Hundreds of different forms of PTMs have been discovered so far and mass spectrometry (MS) has become one of the most important tools to analyze PTMs. With the development of new methods in sample preparation, advances in MS instrumentation, and inventions of new bioinformatic tools, MS-based methods can identify most forms of PTMs, precisely locate the site of modification, sensitively and accurately quantify the changes of modification levels from various of biological samples. This chapter will summarize generic procedures to analyze PTMs with MS methods and provide an update with the latest development in sample preparation, MS analysis, and database search that advanced the way for PTM analysis, improving our knowledge of PTMs and understanding of their roles in various diseases.

Keywords:  Bottom-up; Database search; Mass spectrometry; Post-translational modification; Sample preparation; Top-down

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_1
EMBO Rep. 2026 Aug 07.

Mannose supports lung cancer metabolism during glycolytic limitation.

Theresa Haitzmann, Niklas B Thompson, Hardik Shah, Katharina Schindlmaier, Joseph Jelwan, Thomas O Eichmann, Nia G Hammond, Maia G Clare, Robert B Cameron, Brandon Faubert, Katharina Leithner.

Cancer cells frequently show elevated glucose consumption to support proliferation and survival. This led to the assumption that glycolytic inhibitors could be effective in cancer treatment. However, barriers to clinical implementation remain. Adaptive strategies, such as metabolizing alternative nutrients, may play a role. Here, we investigated the use of an understudied sugar, mannose, in lung cancer cells and xenografts. Stable isotope tracing reveals enhanced contribution of mannose to GDP-mannose and GDP-fucose, key glycosylation precursors, upon treatment with the glycolytic inhibitor 2-deoxyglucose (2-DG) or glucose starvation in vitro. Mannose restores the glucose-withdrawal-induced decrease of GDP-mannose and GDP-fucose pools, and partially rescues proliferation upon 2-DG treatment or glucose deprivation. 13C6-mannose infusion in patient-derived xenograft mice reveals a considerable contribution of mannose to GDP-mannose and GDP-fucose in tumors, which is further enhanced by 2-DG. In normal lungs, the pathway is only partially active. Mannose is also shuttled towards glycolysis in lung tumors in vivo and glucose-deprived cells in vitro. In conclusion, mannose utilization for glycosylation precursor synthesis represents an adaptive strategy in lung cancer cells under metabolic stress.

DOI: https://doi.org/10.1038/s44319-026-00874-6
Methods Mol Biol. 2026 ;3018 155-168

Enrichment and Metaproteomic Analysis of Lysine Acetylation in Fecal Microbiome Samples.

Xu Zhang, Zhibin Ning, Daniel Figeys.

  Among the various posttranslational modifications (PTMs) found in microbiome samples, lysine acetylation is known to be abundant and plays an important role in regulating microbial short-chain fatty acid (SCFA) metabolism. The latter is a crucial microbiome function that significantly impacts human intestinal health. This chapter describes a detailed protocol for lysine acetylomic profiling of microbial proteins in human fecal microbiome samples. The protocol consists of stool sample preprocessing, microbiome protein extraction and digestion, immunoaffinity enrichment of lysine acetylated peptides, and high-resolution mass spectrometry analysis for the identification and quantification of lysine-acetylated proteins.

Keywords:  Bioinformatics workflow; Immunoaffinity enrichment; Lysine acetylation; Metaproteomics; Microbiome

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_10
Nat Metab. 2026 Aug 05.

Palmitate promotes liver metastases by decreasing neutrophil antitumour behaviour.

Anke Vandekeere, Juan Fernández-García, Yiming Peng-Winkler, Miguel Martín Pérez, Ming Liu, Sara Isabel Fernandes, Janine Theile, Philipp Willnow, Sanne Verheul, Gitte Zels, Anirudh Pabba, Christine Desmedt, Cornelis Verhoef, Patricia Altea-Manzano, Salvador Aznar Benitah, Ilaria Elia, Ilaria Malanchi, Sarah-Maria Fendt.

Liver metastases are frequent and challenging to treat owing to the liver's metabolically active and immune-tolerant environment. However, how cancer cells exploit nutrient availability in the liver to evade immune surveillance remains unknown. Here we show that cancer cells use the palmitate availability in the liver to impair the neutrophil antitumour function. Mechanistically, we find that breast and colorectal cancer cells metastasizing to the liver, but not the lung, require the palmitoyltransferase 17 (DHHC17, gene name ZDHHC17) to stabilize laminin-511 enabling its secretion. In turn, neutrophils in the liver metastasis environment respond to laminin-511 by decreasing their cancer cell-killing capacity. Consistently, silencing ZDHHC17 in cancer cells decreases liver metastases only in the presence of neutrophils, while metastasis growth is restored in ZDHHC17-silenced metastases upon injection of laminin-511 or inhibition of neutrophil degranulation. Taken together, we find that liver palmitate not only supports tumour intrinsic processes but also enables immune evasion.

DOI: https://doi.org/10.1038/s42255-026-01582-0
Biochem Pharmacol. 2026 Aug 06. pii: S0006-2952(26)00665-9. [Epub ahead of print] 118326

Beyond bar charts: A practical guide to small sample pharmacological data.

Nancy A Sachs, Michael J Marino.

  In pharmacology and experimental biology, datasets are frequently limited in size (n < 20) due to the high costs and ethical considerations of data acquisition. In these "small-n" scenarios, estimating population parameters is challenging because single observations can disproportionately influence results. This creates a dilemma: while identifying anomalous observations is essential for reliable interpretation, ad-hoc data removal risks introducing investigator bias. Furthermore, many traditional outlier detection methods are optimized for large datasets and perform poorly at sample sizes typical of bench science. This manuscript provides a structured, logical workflow for exploratory data analysis (EDA) and objective outlier management tailored for the biological researcher. We introduce a "Visual Trio" framework incorporating Tukey boxplots, overlaid scatter plots, and "Confidence Funnels." This framework is used to assess data distribution and identify potential anomalies. We subsequently demonstrate the application of Dixon's Q-test as an objective diagnostic screen to guide sensitivity analyses in small samples. Furthermore, we address the special case of left-censored assay non-detects through prespecified sensitivity analyses rather than post-hoc exclusion. To support transparency and reproducibility, we provide annotated R code and links to public web resources, enabling scientists to maximize the value of their experimental data while maintaining statistical integrity prior to formal prespecified confirmatory studies.

Keywords:  Data visualization; Dixon’s Qtest; Exploratory data analysis; Outlier detection; Pharmacological data analysis; Reproducibility; Small sample size

DOI:  https://doi.org/10.1016/j.bcp.2026.118326
Crit Rev Anal Chem. 2026 Aug 07. 1-22

Fit-for-purpose analytics for cannabis: identification, quantification, and standardization of cannabinoids, flavonoids and terpenes.

Eman Shawky, Lutfun Nahar, Satyajit D Sarker, Dina A Selim.

  Cannabis-based products used in medicine and wellness are chemically complex mixtures of cannabinoids, terpenes, flavonoids (including cannflavins), and degradation products whose profiles can shift substantially with cultivar genetics, cultivation conditions, extraction workflows, formulation choices, and storage history. This field-to-field and lab-to-lab variability, compounded by inconsistent analytical practices and uneven validation standards, can undermine potency labeling, cross-product comparability, and the interpretation of clinical and pharmacological findings. This review critically summarizes current quantitative and fingerprint-based strategies for measuring cannabinoids and related constituents across major product types (flowers/biomass, oils, edibles, and vape products) and proposes a practical "field-to-result" framework spanning sampling, method selection, validation, and lifecycle quality control. Drawing on peer-reviewed literature and metrological guidance, we evaluate LC-UV/LC-MS/MS, GC-FID/GC-MS, quantitative and structural NMR, and selected spectroscopic and aerosol approaches, with emphasis on isotope-dilution concepts, calibration design, matrix effects, validation criteria, and chemometric tools for multivariate fingerprints. The evidence is synthesized to map analyte classes and matrices to fit-for-purpose platforms and sample preparation schemes, highlight best practices for internal standards and matrix-matched calibration, and summarize commonly applied acceptance expectations for accuracy and precision. Strategies to reduce or correct matrix effects are detailed, and the role of high-dimensional fingerprints is explained as a complement to targeted potency panels for identity confirmation, process drift monitoring, lot release decisions, and traceability. Overall, combining validated potency assays with statistically governed fingerprinting offers a pragmatic route to harmonize cannabinoid testing, improve data integrity, and better support future phytomedicine research, clinical translation, and regulatory oversight.

Keywords:  Cannabinoids; Cannabis sativa; chemometrics; flavonoids; terpenes

DOI:  https://doi.org/10.1080/10408347.2026.2714459
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Aug 03. pii: S1570-0232(26)00331-4. [Epub ahead of print]1282 125242

Quantifying the aromatic amino acid metabolome: UPLC-MS/MS analysis of aromatic amino acids and their host and co-metabolites in plasma.

Marlene Thaitumu, Domniki Gallou, Olga Begou, Georgios Theodoridis, Helen Gika.

  Aromatic amino acids (AAAs), tryptophan, phenylalanine, and tyrosine along with their pathway metabolites have been implicated in the pathogenesis of diseases ranging from cardiovascular, neurological, inflammatory, and cancer diseases, among others. As such, the measurement of the primary AAAs, their host pathway metabolites, and microbiome derived co-metabolites in blood can provide a sensitive reflection of systemic health. The aim of the study was to develop a method for the quantification of 17 metabolites, the three AAAs and various of their metabolites in plasma using a high-throughput ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. The method demonstrated a dynamic range (1 to 16,700 ng/mL), with detection limits (LOD) as low as 0.05 ng/mL. Quantification limits ranged from 3 to 5019 ng/mL (LLOQ) and up to 16,700 ng/mL (ULOQ). Recovery at LQC, MQC, and HQC was satisfactory and consistent across most metabolites, with significant matrix effects observed only for 4-ethylphenol sulfate. Furthermore, intra and inter-day accuracy and precision met all acceptance criteria at all quality control concentrations for most of the metabolites. Measurement of NIST SRM 1950 showcased the method's accuracy for most of the metabolites. Finally, the method was applied on the analysis of plasma samples from 55 individuals (13 males and 42 females) providing information on AAAs and their pathway metabolites relevant concentrations in human plasma.

Keywords:  Aromatic amino acids pathways; LC–MS/MS; Metabolites; Microbial-host; Phenylalanine; Tryptophan; Tyrosine

DOI:  https://doi.org/10.1016/j.jchromb.2026.125242
Methods Mol Biol. 2026 ;3018 139-153

Mapping Cellular Protein Lysine Methylation Using Targeted-Mass Spectrometry.

Anand Chopra, Matthew Hoekstra, William G Willmore, Kyle K Biggar.

  Lysine methylation is a dynamic posttranslation modification (PTM) involved in the regulation of numerous biological processes. Depending on the context, site- and state-specific lysine methylation has been shown to regulate key features of proteins, such as stability, activity, and interactions. Assessing site- and state-specific lysine methylation within the proteome is difficult without access to antibodies that are able to detect the modification site. As the commercially available options for reliable antibodies that are specific to a methylation site and state are limited, mass spectrometry (MS)-based methods are more appealing as they enable detection of site- and state-specific methylation events with broad applicability. Here, we describe the use of immunoprecipitation and targeted-MS to detect and perform relative quantification of site-specific lysine methylation events occurring on endogenous proteins of interest. As an example, detection and relative quantification of hypoxia-responsive PGC1α-K224me2 and RNF20-K610me3/K614me3 lysine methylation, occurring in hypoxia-treated human colorectal cancer cells, are described.

Keywords:  Hypoxia; Lysine methylation; Parallel reaction monitoring; Posttranslational modification; Targeted-mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_9
Methods Mol Biol. 2026 ;3018 211-225

Profiling O-Acetylation in Sialoglycans Using MALDI-TOF and LC-MS/MS with Methylamidation and Permethylation Derivatization.

Zhaoguan Wu, Isabelle Sirois.

  O-acetylation of sialoglycans plays a critical role in modulating the structural and functional properties of glycoproteins, with implications in immune response, cell signaling, and disease pathology. Among sialic acids, N-acetylneuraminic acid (Neu5Ac) and its derivatives have garnered attention as biomarkers for various cancers and inflammatory conditions due to their terminal positioning on glycan chains and their susceptibility to biochemical modification. Despite their biological relevance, comprehensive analysis of O-acetylation patterns remains challenging due to their chemical lability and loss during conventional glycomic workflows, such as permethylation. This protocol outlines a robust workflow combining methylamidation and permethylation with porous graphitic carbon (PGC)-based purification and high-resolution MALDI-MS and LC-MS/MS to analyze O-acetylation in serum/plasma-derived N-sialoglycans. We demonstrate sensitive and reproducible profiling of sialylated glycan structures. This method enables deeper insight into glycan modification dynamics and can be broadly applied to biomarker discovery and comparative glycomics.

Keywords:  Cancer biomarkers; Glycomics; LC–MS/MS; MALDI–MS; Methylamidation; N-acetylneuraminic acid; O-acetylation; PGC purification; Posttranslational modification; Sialoglycans

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_14
Bioanalysis. 2026 Aug 03. 1-10

Development and validation of a sensitive HPLC-MS/MS method for the analysis of semaglutide in human plasma.

Christopher Tudan, Joel Stickling, Emily Northwick Darden, Lauren Devine, Shane Needham.

   BACKGROUND: Semaglutide is a human glucagon-like peptide-1 (GLP-1) peptide analog. To date, the literature does not report a validated HPLC-MS/MS assay using a triple-quadrupole mass spectrometer for the quantitation of semaglutide in human plasma in support of pharmacokinetics. A robust, sensitive HPLC--MS/MS assay supporting this class of compounds, is provided and full validation demonstrated.
METHODS AND RESULTS: A triple-quadrupole HPLC-MS/MS method has been developed and fully validated for the quantitation of semaglutide in human plasma, using a stable isotope labeled internal standard and an effective, efficient protein precipitation sample extraction. The method has a sensitive analytical range of 5 ng/mL to 200 ng/mL (and to 1000 ng/mL with a dilution QC) and exhibits excellent accuracy (92.7% to 109.4%) and precision of <10.6%. This highly selective method provided high recovery of the analyte, with minimal matrix effects.
CONCLUSION: A robust and simple HPLC-MS/MS method to quantify semaglutide in human plasma has been fully validated in accordance with the 2022 FDA M10 guidance. This method is suitable for implementation in clinical studies within the validated analytical range and stability conditions and can be extended to the bioanalysis of other GLP-1 peptide-based therapeutics with similar structural and physiochemical properties.

Keywords:  GLP-1; HPLC-MS/MS; ICH M10; method validation; peptides; semaglutide

DOI:  https://doi.org/10.1080/17576180.2026.2712160
Methods Mol Biol. 2026 ;3018 185-198

Combining SDS-PAGE with Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics Analysis of Intact Histone Proteoforms.

Fei Fang, Liangliang Sun.

  Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial posttranslational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates histone proteins (H1, H2, H3, and H4) based on their molecular weight, while capillary zone electrophoresis (CZE) provides additional separation of proteoforms of each histone protein based on their electrophoretic mobility, which is affected by PTMs, e.g., acetylation and phosphorylation. Here, we describe the combination of SDS-PAGE-based protein fractionation with CZE-tandem MS (MS/MS) for high-resolution characterization of histone proteoforms. Over 200 histone proteoforms from a commercial calf thymus histone sample were identified with good reproducibility.

Keywords:  Capillary electrophoresis-mass spectrometry; Histone proteoform; Posttranslational modifications; SDS-PAGE; Top-down proteomics

DOI:  https://doi.org/10.1007/978-1-0716-5166-7_12
Biochim Biophys Acta Mol Cell Biol Lipids. 2026 Aug 07. pii: S1388-1981(26)00051-X. [Epub ahead of print] 159765

Remodeling of lipid metabolism orchestrates human adipose-derived mesenchymal stem cells osteodifferentiation.

Daniela S C Bispo, Jennifer H Haggarty, Inês Graça, Brian J Goodfellow, João Rodrigues, João F Mano, Mariana B Oliveira, Penelope M Tsimbouri, Matthew J Dalby, Ana M Gil.

  Mesenchymal stem cell (MSC) osteodifferentiation involves adaptations of lipid metabolism but the specific regulatory roles of lipid species remain underexplored. We use a global metabolomics approach, comprising LC-MS lipidomics and NMR metabotolomics, to identify mechanistic features of human adipose-derived MSC (hAMSC) osteodifferentiation and new markers of osteogenic progression. Results show that, upon differentiation, cellular metabolism progresses through an early osteocommitment phase (day 7) followed by active osteodifferentiation (day 21), with underlying proliferation contributing towards membrane remodeling, oxidative stress protection and lipid-supported energy/signaling processes. At day 7, differentiating cells exhibit coordinated lipid remodeling and activation of the phosphocreatine (PCr)-creatine (Cr) axis. This leads to accumulation of phosphocholine, PCr and sphingomyelin, in preparation for mineralization and extracellular vesicle formation. Concomitantly, calcium oscillatory signaling and active purinergic metabolism are predicted. Other early features persist until day 21, including increased polyunsaturated fatty acids (PUFAs)-rich plasmalogen levels, enhanced endogenous substrate mobilization and metabolic autonomy. At this later stage, accumulation of storage lipids occurs, likely contributing to ω-3/ω-6 balance. FA regulation seems to be accompanied by a shift from TCA/OXPHOS towards β-oxidation and PCr hydrolysis, as predominant energy sources. Late-stage osteodifferentiation is also characterized by inhibition of pathways associated with undifferentiated state maintenance, while several upstream regulators emerge as pro-osteogenic. The resulting metabolic framework enables actionable monitoring and rational optimization of bone tissue engineering strategies.

Keywords:  Lipidomics; Lipids; Mass spectrometry; Mesenchymal stem cells; Metabolic markers; Osteogenic differentiation

DOI:  https://doi.org/10.1016/j.bbalip.2026.159765
Clin Chem Lab Med. 2026 Aug 06.

An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of meropenem in human serum and plasma.

Rupert Schmid, Judith Taibon, Neeraj Singh, Carina Schuster, Arber Rexhaj, Michael Vogeser, Friederike Bauland, Andrea Geistanger, Elie Fux.

   OBJECTIVES: Therapeutic drug monitoring (TDM) of meropenem is critical for optimizing clinical efficacy and minimizing toxicity. We describe the validation of an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) candidate reference measurement procedure (RMP) for the quantification of meropenem in human serum and plasma.
METHODS: A high-purity meropenem standard was characterized using an in-house quantitative nuclear magnetic resonance protocol. This established the absolute content and ensured an unbroken chain of metrological traceability to the International System of Units. Samples were prepared via incubation with a stable isotope-labeled internal standard followed by protein precipitation. Analysis was performed using LC-MS/MS with electrospray ionization. The method was validated to assess selectivity, matrix effects, precision, accuracy, and measurement uncertainty according to international metrological guidelines.
RESULTS: The candidate RMP demonstrated high selectivity across a linear range of 0.400-120 μg/mL and was unaffected by matrix interferences. Intermediate precision was <3.8 % for spiked serum and <4.3 % for native patient pools; repeatability ranged from 1.9 to 3.1 %. Mean bias ranged from -5.1 to 2.4 % across all serum and plasma levels, including dilution integrity assessments. Expanded measurement uncertainty (k=2) for target value assignment (n=6) was 2.6-4.3 %, while uncertainty for single measurements ranged from 6.0 to 8.8 %.
CONCLUSIONS: The proposed method fulfills all analytical performance specifications required for a candidate RMP. By establishing a direct link from individual patient samples to primary reference materials, this approach provides a robust foundation for the global standardization of routine meropenem assays.

Keywords:  SI units; isotope dilution-liquid chromatography-tandem mass spectrometry; meropenem; quantitative nuclear magnetic resonance characterization; reference measurement procedure

DOI:  https://doi.org/10.1515/cclm-2026-0428
Toxicol In Vitro. 2026 Aug 01. pii: S0887-2333(26)00098-6. [Epub ahead of print]117 106290

A targeted lipidomics workflow for high-throughput phenotyping and mechanism-based in vitro hepatotoxicity studies.

Tzu-Hsin Yen, Chander K Negi, Courtney Sakolish, Erin S Baker, Ivan Rusyn.

  Animal-based toxicity testing is limited in throughput and mechanistic characterization, underscoring the need for cell-based approaches. Incorporating molecular readouts, such as lipidomics, can enhance the translational relevance of cell-based assays. Because untargeted lipidomic analyses are time-consuming and can require large sample volumes, a targeted workflow was used in this study to characterize drug-induced liver injury-related lipid responses in primary hepatocytes from multiple species. Multiple reaction monitoring was applied in a targeted lipidomics workflow by coupling liquid-chromatography with triple quadrupole mass spectrometry (LC-MS/MS) for 148 lipid targets across 3 categories and 11 classes. We used primary hepatocytes from human, rat, monkey, and dog cultured in 96-well plates and exposed to chlorpromazine (1-30 μM), bosentan (1-200 μM), and fialuridine (1-100 μM) from culture day 4 to 8. Chlorpromazine induced the most pronounced lipid alterations in human, monkey, and rat hepatocytes, while responses in dog hepatocytes were minimal, with human cells showing a dose-dependent trend up to 10 μM. Bosentan induced dose-dependent lipid changes across species, strongest in human hepatocytes, whereas fialuridine caused limited lipid alterations. Overall, lipidomic effects closely aligned with conventional toxicity markers, reflecting compound and species-specific hepatotoxic sensitivity. The targeted lipidomics workflow provided mechanism-informed phenotyping of chemical-induced lipid responses in cultured hepatocytes. Moreover, it provided a framework for mechanistic evaluation, supported cross-species comparison, and complemented in vitro hepatotoxicity assessments.

Keywords:  Cross species; DILI; In vitro Lipidomics; NAMS

DOI:  https://doi.org/10.1016/j.tiv.2026.106290
bioRxiv. 2026 Jul 01. pii: 2026.06.30.734970. [Epub ahead of print]

NNMT Loss Drives Cancer Progression by enhancing SAM availability for mTORC1 Signaling and Chromatin Methylation.

Qu Deng, Erick Mitchell-Velásquez, Sharan Venkatesh, Rahul Mannan, Hyein Cho, Mohammed Alhusayan, Aqsa Yashfeen, Ramakrishnan Natesan, Natarajan V Bhanu, Radha Paturu, Javed Siddique, Rohit Mehra, Sooryanarayana Varambally, Benjamin Garcia, David Feldser, Priti Lal, Arul M Chinnaiyan, Irfan A Asangani.

  Aberrant epigenetic reprogramming together with dysregulated mTOR signaling are hallmarks of cancer, where altered chromatin methylation and nutrient-sensing pathways cooperate to drive tumor progression. S-adenosylmethionine (SAM), the universal methyl donor, is essential for these processes, yet how tumors sustain elevated SAM availability to support oncogenic transmethylation reactions remains poorly defined. Here, using prostate cancer (PCa) as a model system, we identify nicotinamide N-methyltransferase (NNMT) as a critical metabolic-epigenetic regulator and tumor suppressor. Using a prostate-specific Nnmt knockout mouse model, we demonstrate that NNMT loss accelerates PCa progression, particularly in the context of Pten deletion, resulting in infiltrating carcinoma and reduced survival. Mechanistically, NNMT functions as a "SAM-sink," and its loss increases intracellular SAM abundance, thereby activating mTORC1 signaling through SAMTOR-dependent sensing and broadly enhancing chromatin methylation. In human PCa, recurrent genomic deletions of NNMT occur in up to 7% of cases, and NNMT protein expression is largely absent in primary tumors and metastases. NNMT-deficient PCa cells exhibit elevated SAM:SAH ratios, increased histone methylation, and heightened mTORC1 activity, enabling sustained tumor growth even under dietary methionine-restriction (MR). Notably, combined MR and pharmacologic mTORC1 inhibition synergistically suppresses the growth of NNMT-deficient tumors, revealing a previously unrecognized therapeutic vulnerability. Collectively, these findings establish NNMT as a key tumor suppressor that constrains SAM-driven epigenetic and signaling programs in PCa and suggest a rational, diet-based therapeutic strategy for advanced cancers with NNMT loss.

Keywords:  SAM-sink; epigenetics; mTOR signaling; metabolism; prostate cancer; therapeutics; transmethylation

DOI:  https://doi.org/10.64898/2026.06.30.734970
Curr Opin Biotechnol. 2026 Aug 05. pii: S0958-1669(26)00122-9. [Epub ahead of print]101 103557

Computational metabolomics at scale: from open data to insight.

Ewy A Mathé, Justin Jj van der Hooft, Haley Chatelaine, Louis-Félix Nothias, Stacey N Reinke, Juan Antonio Vizcaíno, Egon L Willighagen, Timothy Md Ebbels, Soha Hassoun.

Metabolomics data are currently generated at scale thanks to the evolution of technologies that have led to marked improvements in the number of metabolites detected, spanning all chemical classes. These data are increasingly submitted to public repositories for data reuse, integration, and interpretation. Despite the availability of public resources and associated computational tools, the field still lacks a widely adopted, consistent data and analytics infrastructure capable of transforming this wealth of information into scientific insight. Indeed, the metabolomics field is just now scratching the surface of being able to harness the power of new computational technologies. In this review, we summarize discussions from the "Dagstuhl-Seminar 24181 Computational Metabolomics: Towards Molecules, Models, and their Meaning" with a focus on public data availability, open data standards, data and knowledge integration, and education. Our goal is to raise awareness and adoption of the latest open science resources while highlighting key areas needing further development.

DOI: https://doi.org/10.1016/j.copbio.2026.103557