bims-mascan 2026-06-28 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2026–06–28
eighteen papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Limited impact of column chemistry and length on proteome coverage under high-speed DIA.
A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High Performance Proteomics Analysis.
Integrated Multiomics Enabled by Sequential Extraction for Comprehensive Molecular Profiling of Small Extracellular Vesicles.
DiaReport: Reproducible workflow for differential expression analysis and interactive reporting in DIA-based proteomics.
Practical Workflow for Building Local Mass Spectral Libraries for Untargeted Metabolomics.
Comparative Evaluation of DDA- and DIA-Based Proteomic Workflows in Beryllium-Related Lung Disease.
Development of a Xylene-Free Sample Preparation Protocol for Quantitative Proteomics of Clinically Relevant Formaldehyde-Fixed Paraffin-Embedded Needle Biopsy Samples.
QproMS: a web application for label-free proteomic data analysis.
Evaluation of TIMS-derived peptide mobility measurements: calibration strategy and MS1/MS2 discrepancies.
MS-Based Crevicular Fluid Proteomics for the Study of Periodontal and Peri-Implant Conditions: A Systematic Review.
Development of Mass Spectrometry-Based SCFA Analysis Methods in Diverse Samples for Microbiome Research.
Isotope-Coded Derivatization Enables Accurate LC-MS/MS Quantification of Urinary THC-COOH without Isotope-Labeled Cannabinoid Standards.
LC-MS assay for quantifying ornithine decarboxylase activity in the biological matrix and cultured cells using stable isotope‑labeled ornithine.
Systematic characterisation of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry.
Benchmarking Lipid Quantitation from Skin Swab Sebum-Rich Samples to Establish Mass Spectrometry-Based Diagnostic Methodology.
Column coupling for chiral LC-MS separation resolves triacylglycerol enantiomers and regioisomers in complex samples without flow recycling.
Depletion-Free Automated Enrichment of Serum Glycopeptides for High-Throughput Clinical Glycoproteomics.
Optimized Digestion Conditions for Membrane Protein Footprinting and Mass Spectrometry Analysis.

Mol Cell Proteomics. 2026 Jun 22. pii: S1535-9476(26)00105-2. [Epub ahead of print] 101609

Limited impact of column chemistry and length on proteome coverage under high-speed DIA.

Alicia-Sophie Schebesta, Kathrin Korff, Ericka C M Itang, Vincent Albrecht, Philipp E Geyer, Johannes B Mueller-Reif.

The evolution of mass spectrometry (MS)-based proteomics has been driven by continuous technological advances in sample preparation, liquid-phase separations, instrumentation, and data acquisition. Chromatographic performance has been recognized as a contributing factor to identification depth, particularly on earlier-generation MS platforms. Recent advances in MS sampling speed and sensitivity now raise the question of how strongly chromatographic quality continues to determine overall proteome coverage. We investigate how column chemistry and length influence proteome coverage and chromatographic selectivity under modern DIA conditions, and whether traditional optimization priorities still apply. Spanning a matrix of experiments with five distinct stationary phases, including C18 chemistries, C8, and Phenyl-Hexyl, across eight column lengths (40-140 mm), we evaluate protein identification performance using data-independent acquisition (DIA) on the Orbitrap Astral mass spectrometer. Despite differences in stationary-phase chemistry and column length, we observed remarkably convergent proteome coverage metrics. All C18 and C8 phases consistently achieved over 150,000 precursor- and approximately 9,000 protein group identifications, regardless of column length variations. While retention fingerprints persisted across chemistries, these chromatographic differences did not translate into meaningful variations in proteome coverage under high-speed acquisition conditions at 200 Hz. Within the range of modern sub-2 μm reversed-phase materials tested, identification depth showed limited dependence on column chemistry and length, suggesting that for state-of-the-art stationary phases, method development priorities may increasingly favor operational robustness, throughput, and reproducibility over traditional separation optimization.

DOI: https://doi.org/10.1016/j.mcpro.2026.101609
Mol Cell Proteomics. 2026 Jun 22. pii: S1535-9476(26)00104-0. [Epub ahead of print] 101608

A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High Performance Proteomics Analysis.

Leonard C Rorrer, Liulin Deng, Lauren Royer, Isabel Uribe, Benjamin C Orsburn, Oliver Bernhardt, Tejas Gandhi, Lukas Reiter, Daniel DeBord.

Here we present a novel data independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation (SLIM) technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. PAMAF mode increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering, ion losses are avoided while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. Additionally, by accumulating ions while the previous packet of ions is being analyzed, the PAMAF mode achieves ∼100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed ∼6x more protein group identifications compared to a standard data-dependent acquisition (DDA) analysis without HRIM on the same QTOF instrument, and to >100x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by DDA. Additionally, since precursor isolation in PAMAF mode is size-based rather than m/z-based, coeluting isobars and isomers can be resolved prior to fragmentation, eliminating chimeric spectra that compromise identification accuracy. We also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity termed DIA-PAMAF mode, which enabled the detection of over 8,000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics with the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics.

DOI: https://doi.org/10.1016/j.mcpro.2026.101608
Anal Chem. 2026 Jun 22.

Integrated Multiomics Enabled by Sequential Extraction for Comprehensive Molecular Profiling of Small Extracellular Vesicles.

Andrew J Perciaccante, Holden T Rogers, Yanlong Zhu, Aditi Barnwal, David Inman, Man-Di Wang, Song Jin, Suzanne M Ponik, Ying Ge.

Small extracellular vesicles (sEVs) are membrane-bound particles whose protein, lipid, and metabolite cargo reflects the molecular state of their cells of origin, making them attractive targets for biomarker discovery and therapeutic development. However, comprehensive characterization of sEVs remains challenging due to the extremely limited material available. Here, we present an integrated mass spectrometry-based multiomics platform for simultaneous characterization of lipids, metabolites, and proteins from a single sEV sample enabled by sequential extraction, maximizing sample utilization. To enhance molecular coverage and analytical depth, the platform combines iterative tandem mass spectrometry for improved small-molecule fragmentation and nanoflow proteomics with data-independent acquisition. We achieved deep and reproducible multiomic characterization of proteins, lipids, and metabolites using 10 million sEVs. We further demonstrated the compatibility of our multiomics platform with sEVs isolated from plasma by ultracentrifugation, size exclusion chromatography with ultrafiltration, and polymer precipitation, revealing purification-dependent differences in molecular profiles associated with trade-offs in yield and purity of sEVs. By enabling integrated multiomics from the same sample, this strategy addresses a key challenge in low-input sEV analysis and establishes a robust analytical foundation for synergistic biomarker discovery and therapeutic applications.

DOI: https://doi.org/10.1021/acs.analchem.6c01280
Bioinformatics. 2026 Jun 22. pii: btag435. [Epub ahead of print]

DiaReport: Reproducible workflow for differential expression analysis and interactive reporting in DIA-based proteomics.

Andrea Argentini, Esperanza Fernández, Jarne Pauwels, Kris Gevaert.

MOTIVATION: Data-independent acquisition (DIA) has become the preferred data acquisition method for mass spectrometry-based proteomics, yet, reproducible workflows for differential expression (DE) analysis and results reporting remain limited. We present DiaReport, an R package that performs precursor- and protein-level DE analysis from DIA-NN output using MSqRob and QFeatures, while generating high-quality, interactive HTML reports through Quarto. DiaReport integrates precursor data, filtering of missing values, normalization, protein summarization and statistical modeling within a single function, supporting both simple pairwise as well as complex experimental designs. The package provides structured outputs and configuration files to ensure computational reproducibility across different studies. To accommodate diverse research needs, DiaReport includes multiple reporting templates tailored to different proteomic applications. Applying DiaReport to an extracellular vesicle (EV) proteomics dataset demonstrates its ability to efficiently analyze DIA data and provide rapid insights into sample quality and protein level differences.
AVAILABILITY: DiaReport is an open-source R package available at https://github.com/Gevaert-Lab/diareport (DOI: 10.5281/zenodo.20120604). The package is platform-independent and distributed under the MIT license. Reports are generated using Quarto and require only standard R dependencies. Detailed documentation, installation guides and usage vignettes are provided within the repository. The interactive HTML reports discussed in this study, including the UPS2 benchmark and EV case study, are archived on Zenodo (DOI: 10.5281/zenodo.20122506 and 10.5281/zenodo.20123378).
SUPPLEMENTARY INFORMATION: Figure S1 (Benchmarking performance of DiaReport); Table S1 (Guidance for missing value filtering strategies); and Table S2 (Indicative runtimes across different cohort sizes and storage configurations) are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btag435
Metabolites. 2026 Jun 12. pii: 412. [Epub ahead of print]16(6):

Practical Workflow for Building Local Mass Spectral Libraries for Untargeted Metabolomics.

Torbjørn Norberg Myhre, Terkel Hansen, Tetiana Lutchyn, Marie Mardal, Terje Vasskog.

  Background: Metabolite identification and annotation remain major bottlenecks in untargeted metabolomics because mass spectral features often lack sufficient specificity. High-confidence annotation requires experimental validation using authentic standards analyzed under matched chromatographic and ionization conditions, providing greater reliability than in silico predictions or database matching alone. This study aimed to develop a practical and scalable workflow for constructing a high-quality mass spectral library using a commercially available analytical standards kit. Methods: A total of 603 metabolites from the MSMLS kit were organized into 42 mixtures, each containing approximately 15 compounds. Mixture design was based on molecular mass and distribution coefficient values, specifically logD at pH 3.1, with a minimum logD spacing of 0.15 to improve chromatographic separation and reduce co-elution. This strategy was used to minimize the total number of injections while maintaining spectral quality. The resulting spectra were evaluated against online spectral resources and in silico fragmentation predictions. A preliminary proof-of-concept analysis was also performed using human serum samples. Results: Using this workflow, 471 metabolites, corresponding to approximately 78% of the standards, were successfully detected and incorporated into the spectral library. Comparison with online resources and in silico fragmentation predictions demonstrated improved spectral quality and reliability. The proof-of-concept serum analysis enabled identification of endogenous metabolites using the constructed library. In addition, the robustness and applicability of the workflow were further supported by a method validation study using metabolites derived from this library. Conclusions: This workflow provides a scalable strategy for constructing mass spectral libraries that balances spectral quality with analytical throughput. By using rational mixture design and authentic standards analyzed under matched experimental conditions, the approach enables substantial metabolite coverage while maintaining data reliability and minimizing experimental effort.

Keywords:  LC-MS/MS; MSMLS; compound discoverer; fragmentation spectra; mass spectral library; mzVault

DOI:  https://doi.org/10.3390/metabo16060412
bioRxiv. 2026 Jun 09. pii: 2026.06.04.730108. [Epub ahead of print]

Comparative Evaluation of DDA- and DIA-Based Proteomic Workflows in Beryllium-Related Lung Disease.

Danielle O Weise, Kanika Gupta, Timothy J Griffin, Pratik D Jagtap, Margaret M Mroz, Reid Wagner, Joshua D Macaluso, S Mehta, Lisa A Maier, L Li, B E Vestal, M Bhargava.

We compared traditional data-dependent acquisition mass spectrometry (DDA-MS) with the increasingly adopted data-independent acquisition (DIA-MS) to evaluate their relative utility for large-scale quantitative biofluid proteomics of lung compartments, specifically paired bronchoalveolar lavage (BAL) cells and bronchoalveolar lavage fluid (BALF). Using beryllium-related granulomatous lung disease as a focused model, we analyzed BALF and BAL cells from beryllium-sensitized (BeS) individuals using both acquisition strategies to assess proteome depth, quantitative completeness, and analytical robustness. In BAL cells, 5,640 proteins were identified by DDA-MS and 5,227 by DIA-MS; however, DIA-MS yielded markedly improved quantitative completeness, with 5,178 proteins (∼99%) quantified across all samples compared with 3,539 (∼63%) quantified by DDA-MS. While 3,397 proteins were quantified by both methods, DIA-MS uniquely quantified 1,781 lower-abundance proteins. Proteins identified by both DIA and DDA-MS approaches revealed pathways associated with granulomatous inflammation, including Toll-like receptor, clathrin-mediated endocytosis, sirtuin, and C-type lectin receptor signaling, whereas DIA-MS resolved additional pathways, such as the complement cascade, coagulation system, and JAK/IL-6-type cytokine signaling. In BALF, although more proteins were identified by DDA-MS than by DIA-MS (2,069 vs 1,742), DIA-MS achieved greater quantitative completeness, with 1,695 proteins quantified across all samples compared with 1,050 using DDA-MS, underscoring its suitability for biomarker-oriented analyses in lung fluid compartments. Together, these results support DIA-MS as a robust and sensitive platform for quantitative lung proteomics and discovery of disease-relevant protein signatures.

DOI: https://doi.org/10.64898/2026.06.04.730108
Proteomes. 2026 Jun 14. pii: 30. [Epub ahead of print]14(2):

Development of a Xylene-Free Sample Preparation Protocol for Quantitative Proteomics of Clinically Relevant Formaldehyde-Fixed Paraffin-Embedded Needle Biopsy Samples.

Gontse Mabuse Moagi, Lívia Beke, Gábor Méhes, Gábor Kecskeméti, Zoltán Szabó, Lilla Turiák, Éva Csősz.

   BACKGROUND: Fresh frozen tissues are considered the gold standard for proteomic analyses due to their superior preservation of protein integrity; however, their use is limited by the logistical and financial requirements of long-term cold storage. Formaldehyde-fixed paraffin-embedded (FFPE) tissues provide a practical alternative, owing to their stability and widespread availability in clinical settings. A critical step in FFPE proteomics is deparaffinization, which traditionally relies on organic solvents such as xylene, along with the efficient reversal of formaldehyde-induced crosslinks.
METHODS: In this study, we evaluated multiple FFPE protein extraction and digestion workflows including chaotropic, surfactant-based, and detergent-free approaches in combination with xylene-free deparaffinization strategies, using label-free data-independent acquisition (DIA) LC-MS/MS.
RESULTS: Among the tested methods, a chaotropic, reductant, and surfactant-free in-solution digestion workflow demonstrated robust protein and peptide recovery. A modified version of this protocol further improved peptide coverage while maintaining comparable protein depth. The applicability of the optimized workflow was assessed using FFPE needle biopsy samples from control, hepatic steatosis, and liver fibrosis groups. Exploratory proteomic patterns were observed across conditions, with hepatic steatosis associated with early activation of stress-response pathways, while fibrosis showed evidence suggesting altered lipid metabolism.
CONCLUSIONS: Overall, this study presents a simple, xylene-free, and MS-compatible workflow for FFPE proteomics that is suitable for low-input clinical samples and may support broader application of archival tissues in proteomic research.

Keywords:  DIA; FFPE; HAIR; mass spectrometry; needle biopsy; xylene-free

DOI:  https://doi.org/10.3390/proteomes14020030
Bioinform Adv. 2026 ;6(1): vbag158

QproMS: a web application for label-free proteomic data analysis.

Fabio Bedin, Giorgia Cucina, Giampaolo Martinello, Stefano Rizzieri, Andrea Graziadei, Alessandro Cuomo.

Motivation: Proteomics has experienced substantial growth in methods and data analysis approaches, with the development of new data-dependent (DDA) and data-independent acquisition (DIA) workflow and several search engine algorithms and software packages. Each of these workflows has its unique data analysis package that performs data reduction, missing value imputation, statistical testing, and visualization. Often, these tools are designed for expert users.
Results: We present Quantitative Proteomics Made Simple (QProMS), a user-friendly, search engine-agnostic data analysis and visualization pipeline. QProMS guides the user through data analysis and statistical testing in a graphical interface. Statistical tests rely on established R functions and are compatible with all types of label-free quantification experiments. The pipeline recapitulates features from different available software packages and introduces mixed imputation, an improved framework for handling missing values that does not rely on machine learning. QProMS can also perform interaction analyses based on gene ontology, or by querying protein-protein interaction databases. All figures in QProMS are interactive, allowing for investigation of individual proteins of interest before export. The analysis can be saved in a standalone report. QProMS provides a platform for reproducible proteomic data analysis for novice and experienced users, enabling state-of-the-art data analysis of a wide variety of label-free proteomic workflows ranging from global proteome profiling to targeted methods such as proximity labeling.
Availability and implementation: QProMS is accessible as a web server hosted at https://shiny.bioserver.ieo.it/app/qproms or can be run locally as a standalone Shiny application with the code and instructions provided at https://github.com/ieoresearch/QProMS. The application may also be run locally by installing it as a library/package and running a single command as described in the README. Code to generate benchmarking is available at https://github.com/grandrea/mixed-imputation-benchmark.

DOI: https://doi.org/10.1093/bioadv/vbag158
Anal Bioanal Chem. 2026 Jun 26.

Evaluation of TIMS-derived peptide mobility measurements: calibration strategy and MS1/MS2 discrepancies.

Anh Tran, Tytus Mak, Meghan Burke Harris.

  The widespread adoption of trapped ion mobility coupled to mass spectrometry for proteome analysis necessitates high-quality data measurement of both ion mobility and mass spectra. In this study, the accuracy of ion mobility measurements in the context of mass spectral (MS) library building and searching is evaluated. Key factors influencing measurement accuracy, such as calibration strategy and the timing of acquisition relative to the chromatographic apex, are systematically investigated. A calibration system based on a synthetic peptide mixture is shown to improve calibration performance. Further, reproducibility of MS1- and MS2-associated mobility measurements in data-dependent acquisition (DDA) is analyzed. Overall, this work identifies methods to improve ion mobility measurement accuracy for proteomics analyses using a standardized synthetic peptide mixture.

Keywords:  Ion mobility; Mass spectrometry; Proteomics

DOI:  https://doi.org/10.1007/s00216-026-06633-9
J Proteome Res. 2026 Jun 24.

MS-Based Crevicular Fluid Proteomics for the Study of Periodontal and Peri-Implant Conditions: A Systematic Review.

Laís de Paula Sumback Sivila Souza, Débora Reis Dias, Giuliano Portolese Sversutti Cesar, Débora de Almeida Bianco, Mauricio Guimarães Araújo, Flavia Matarazzo.

  This systematic review aimed to evaluate the current literature on mass spectrometry (MS)-based proteomic analysis of the crevicular fluid in different periodontal and peri-implant conditions, and to summarize methodological differences among studies. A search of electronic databases was conducted and clinical studies using MS-based proteomics in gingival (GCF) and/or peri-implant crevicular fluid (PICF) were considered for inclusion. The findings were synthesized and methodological variations described. A modified QUADOMICS tool was applied for risk of bias assessment. Thirteen studies; five longitudinal and eight cross-sectional were analyzed. Patients ranged from 10 to 190, with 42 to 3070 human proteins identified. Sample preparation and preanalytical procedures differed among studies. Protein identification, characterization and quantification were conducted using different algorithms and computer software against different databases. Different strategies were used to select distinctive proteins. Six studies attempted at biomarker development using different protein selection and validation criteria. While six studies presented moderate quality, seven were considered to be low quality. The present findings emphasize the need for methodological harmonization, including standardized protocols for GCF/PICF collection, harmonized proteomic workflows, multicenter longitudinal validation studies, and targeted mass spectrometry approaches for biomarker verification before they can be translated into the clinical practice.

Keywords:  biomarkers; crevicular fluid; liquid chromatography; mass spectrometry; peri-implantitis; periodontitis

DOI:  https://doi.org/10.1021/acs.jproteome.5c01217
Life (Basel). 2026 Jun 09. pii: 974. [Epub ahead of print]16(6):

Development of Mass Spectrometry-Based SCFA Analysis Methods in Diverse Samples for Microbiome Research.

Chaeeun Park, Md Abdur Rahim, Indrajeet Barman, Hanieh Tajdozian, Youjin Yoon, Sukyung Kim, Mijung Kim, Hoonhee Seo, Ho-Yeon Song.

  With the growing interest in the microbiome, short-chain fatty acids (SCFAs) have emerged as key metabolites due to their critical roles in host physiology, including immune regulation, energy homeostasis, and inflammatory control. As a result, the accurate quantification of SCFAs in various biological samples has become increasingly important. However, reliable and standardized methods for measuring SCFAs across different sample types remain underdeveloped, highlighting the need for methodological refinement. To address this need, we optimized two analytical methods, headspace GC-MS and GC-MS/MS, for SCFA quantification. These techniques were applied to a range of biological matrices, including pure microbial cultures, low-abundance animal liver, animal feces, and standardized simulated human fecal samples. The headspace GC-MS approach enables direct analysis with minimal sample preparation, thereby enhancing throughput and ease of use. In contrast, the GC-MS/MS method, involving methanol extraction, alkaline treatment, and derivatization with MTBSTFA, offers superior sensitivity and precision, making it particularly suitable for small-volume and low-abundance samples. Together, these optimized protocols provide robust, sensitive platforms for profiling SCFAs across diverse biological matrices, facilitating a deeper understanding of microbiome-host interactions and supporting future translational applications.

Keywords:  GC-MS/MS; biological matrices; headspace analysis; microbiome; short-chain fatty acids (SCFAs)

DOI:  https://doi.org/10.3390/life16060974
J Am Soc Mass Spectrom. 2026 Jun 24.

Isotope-Coded Derivatization Enables Accurate LC-MS/MS Quantification of Urinary THC-COOH without Isotope-Labeled Cannabinoid Standards.

Maiko Kusano, Shimba Kawasue, Yu-Ki Sakamoto, Mahiro Yoshida, Taka-Aki Matsuyama.

  Quantitative determination of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in urine is the internationally accepted approach for verifying cannabis use in forensic toxicology. Accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis typically relies on stable isotope-labeled internal standards; however, isotope-labeled cannabinoid reference materials are costly, tightly regulated, and not uniformly accessible across laboratories. We report an isotope-coded derivatization (ICD) strategy that enables isotope-dilution-equivalent quantification of urinary THC-COOH without reliance on commercially available isotope-labeled cannabinoid standards. THC-COOH was derivatized with isopropyl-piperidine carboxylic acid hydrazide (IPPAH) and its deuterated analogue (IPPAH-d6), generating a chemically matched analyte/internal standard pair through parallel derivatization. The derivatives exhibited stable chromatographic coelution and closely matched ionization behavior under positive electrospray ionization. Quantitative LC-MS/MS analysis demonstrated linearity over 1-500 ng/mL (r2 > 0.999), with intra- and interday accuracy of 81.8-108.6% and precision below 10%. Internal standard-normalized matrix factors ranged from 97-109% with minimal variability, indicating effective compensation for matrix effects. This compensation enabled reliable quantification without extensive sample cleanup, allowing a simplified sample preparation workflow compared with conventional methods. Derivatized samples remained stable for at least 72 h in the autosampler without detectable isotopic exchange. This ICD strategy provides a quantitatively reliable alternative to conventional isotope dilution while eliminating dependence on isotope-labeled cannabinoid standards, thereby expanding the accessibility of high-confidence LC-MS/MS quantification and illustrating the broader potential of isotope-coded derivatization in forensic mass spectrometry.

Keywords:  11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC−COOH); isotope-coded derivatization; liquid chromatography-tandem mass spectrometry; Δ9-tetrahydrocannabinol (Δ9-THC)

DOI:  https://doi.org/10.1021/jasms.6c00110
Amino Acids. 2026 Jun 21.

LC-MS assay for quantifying ornithine decarboxylase activity in the biological matrix and cultured cells using stable isotope‑labeled ornithine.

Yuma Shiomi, Naoya Ogawa, Kentaro Takahama, Atsushi Murai, Kyohei Furukawa.

  Polyamines (putrescine, spermidine, and spermine) are essential for animal health and development, and their intracellular levels must be tightly regulated to maintain normal cellular functions. Ornithine decarboxylase (ODC) catalyzes the rate-limiting decarboxylation step in polyamine biosynthesis, and thus accurate assessment of its activity is vital for studies of polyamine metabolism. However, conventional ODC assays rely on radiolabeled substrates and require specialized facilities for radioactive handling. To overcome these limitations, we developed a sensitive and non-radioactive ODC assay using stable isotope-labeled ornithine in combination with liquid chromatography-mass spectrometry (LC-MS). In this protocol, animal tissues (e.g., 0.5 g) or cultured cells (e.g., 2.0 × 106 cells) are homogenized, and cytosolic fractions are prepared by centrifugation. These fractions are incubated with d7-ornithine under 37 °C conditions, and the produced d7-putrescine is derivatized with dansyl chloride and quantified by LC-MS analysis. Reaction samples showed a distinct peak corresponding to d7-putrescine, whereas negative control displayed negligible signals. Moreover, d7-spermidine and d7-spermine were not detectable under these conditions, indicating d7-putrescine production directly reflects ODC activity. We optimized reaction time and substrate concentrations to ensure linearity and precision, and confirmed that the assay responds appropriately to pharmacological inhibition of ODC. Collectively, this protocol provides a practical, sensitive, and non-radioactive method for quantifying ODC activity in both animal tissues and cultured cells, and it offers an accessible tool for polyamine metabolism research.

Keywords:  DFMO; Dansyl chloride; LC–MS; Ornithine decarboxylase; Polyamines; Stable isotope

DOI:  https://doi.org/10.1007/s00726-026-03539-9
Elife. 2026 Jun 25. pii: RP108128. [Epub ahead of print]14

Systematic characterisation of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry.

Hao Jiang, Jimena Druker, James W Wilson, Dalila Bensaddek, Jason R Swedlow, Sonia Rocha, Angus I Lamond.

  We have developed a robust workflow to identify proline hydroxylation sites in proteins, combining hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-liquid chromatography-mass spectrometry (LC-MS) with refining and filtering parameters during data analysis. Using this approach, we have combined data from cell lines treated with either the prolyl hydroxylase (PHD) inhibitor, Roxadustat (FG-4592), or with the proteasome inhibitor MG-132, or with a DMSO control, to identify a total of 4993 and 3247 proline hydroxylation sites, respectively, in HEK293 and RCC4 cells. Of these, 1954 (HEK293) and 1253 (RCC4) high-confidence non-collagen sites were inhibited by FG-4592. Hydroxylated peptides showed consistent characteristics across both datasets, including enrichment in more hydrophilic HILIC fractions and distinct charge and mass distributions compared to unmodified or oxidised peptides. The intensity of the diagnostic hydroxyproline immonium ion varied with MS collision energy, peptide concentration, and adjacent amino acid sequence. Using synthetic peptides, we demonstrate that combining LC retention time with optimised MS parameters enables reliable site identification, even with multiple proline residues present. Proteins with FG-4592-inhibited hydroxylation sites were enriched for roles in RNA metabolism, mRNA splicing, and cell cycle regulation, including the phosphatase 1 regulatory subunit Repo-Man (CDCA2).

Keywords:  HILIC; PHDs; PTM; biochemistry; cell biology; chemical biology; human; mass spectrometry; proline hydroxylation; proteomics

DOI:  https://doi.org/10.7554/eLife.108128
Anal Chem. 2026 Jun 24.

Benchmarking Lipid Quantitation from Skin Swab Sebum-Rich Samples to Establish Mass Spectrometry-Based Diagnostic Methodology.

Minhui Zhu, Caitlin Walton-Doyle, Eleanor Sinclair, Katherine Hollywood, Monty Silverdale, Michael Morris, Perdita Barran.

Skin swab sampling provides an accessible, noninvasive method for collecting lipid-rich material from the skin surface, with growing potential for biomarker discovery and diagnostic testing. However, quantitative sebum analysis remains challenging because of variability in the amount collected and the complexity of the lipid matrix. This study evaluates the robustness of skin swab sampling for lipid analysis and the feasibility of quantifying skin-surface components using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple reaction monitoring was used to assess analytical linearity, matrix effects, and recovery following established protocols for swab collection, storage, and extraction. Robust linearity was observed for both spiked isotope-labeled internal standards (IS) and endogenous lipids across concentrations of 0.15-550 ng/mL (R2 > 0.97). IS recovery varied between lipid classes, with consistent under-recovery. Although IS corrected for analytical variability during extraction and analysis, it could not fully account for differences in the amount of material collected. Endogenous lipids were, therefore, evaluated as normalization markers. Ratios between selected triacylglycerol (TG) species were highly conserved across samples, with coefficients of variation below 15% for TG 45:1/TG 47:1, TG 46:1/TG 48:1, and TG 47:1/TG 48:1. These ratios were stable across individuals and were independent of disease status. The reproducibility of TG recovery and stability of TG ratios suggest that endogenous lipid normalization can mitigate sampling and extraction variability. This approach provides a framework for improving quantitative reliability in skin swab lipidomics and represents a big step toward the development of reproducible mass spectrometry workflows for skin-swat-based biomarker studies.

DOI: https://doi.org/10.1021/acs.analchem.5c08056
Talanta. 2026 Jun 23. pii: S0039-9140(26)00858-1. [Epub ahead of print]311 130202

Column coupling for chiral LC-MS separation resolves triacylglycerol enantiomers and regioisomers in complex samples without flow recycling.

Jenny M Nilsson, David Balgoma, Edvin Dedering Stephansson, Sofia Landström, Curt Pettersson, David Dahlgren, Hans Lennernäs, Mikael Hedeland.

  The positional distribution of fatty acids in the glycerol of triacylglycerols (TGs) gives multiple regioisomeric and enantiomeric species with different absorption, metabolism and tissue distribution. This diversity is further enhanced by the positional isomers of double bonds in these fatty acids which present an analytical challenge. To improve the determination of this plethora of isomers, we coupled chiral liquid chromatography with electrospray ionization high-resolution mass spectrometry. For the separation of enantiomers and regioisomers, we used an amylose tris(3-chloro-4-methylphenylcarbamate) stationary phase (Chiralpak IF-3) and evaluated the key chromatographic parameters: mobile phase composition, column temperature and flow-rate. Acetonitrile/water (98:2, v/v) as mobile phase at 35 °C and 0.15 mL/min was selected for separation of enantiomers and regioisomers. In addition, coupling three columns (750 mm) improved efficiency and resolution for the separation of key enantiomers and regioisomers. In addition, our method discriminated TGs differing in double-bond position and cis/trans configuration, which are difficult to resolve using conventional reversed-phase chromatographic systems. The separation method showed stable retention and reproducible determination of enantiomeric percentages in liver matrices. The application of the method revealed the stereochemical TG diversity in rat liver extracts that is unresolved by conventional RP chromatography. Furthermore, it enabled detection of differences in enantiomeric distribution following exposure to doxorubicin. This highlights the importance of resolving TG isomers in biological samples. In conclusion, this work reports a robust MS-compatible lipidomics chiral platform for stereospecific TG analysis providing an analytical foundation for future investigation of lipid remodelling and de novo lipogenesis at the stereochemical level.

Keywords:  Chiral LC-MS; Chiralpak IF-3; Double-bond isomers; Enantiomeric separation; Regioisomeric separation; Stereospecific analysis; Triacylglycerols

DOI:  https://doi.org/10.1016/j.talanta.2026.130202
Mol Cell Proteomics. 2026 Jun 25. pii: S1535-9476(26)00107-6. [Epub ahead of print] 101611

Depletion-Free Automated Enrichment of Serum Glycopeptides for High-Throughput Clinical Glycoproteomics.

Jung Hoon Choi, Geul Bang, Jong Hyun Seol, Seong Woo Choi, Su-Min Lee, Bao Tan Nguyen, Seungjin Na, Cheol-Jung Lee, Heeyoun Hwang, Jinsoo Chung, Sang-Jae Park, Sang Myoung Woo, Hye Gwang Jeong, Jin Young Kim.

  High-throughput and reproducible enrichment of N-glycopeptides remains a major bottleneck in large-scale and clinical glycoproteomics, despite substantial advances in mass spectrometry. Here, we present a depletion-free, high-throughput N-glycopeptide enrichment workflow (3kMAX) that combines 3 kDa molecular weight cut-off (MWCO) filtration with positive-pressure-driven mixed anion exchange (MAX) enrichment in a 96-well format. This platform enables parallel processing of up to 96 samples with minimal operator intervention, improving analytical throughput, robustness and inter-batch reproducibility. Systematic evaluation using human serum demonstrated that the combined workflow achieved superior glycopeptide selectivity and identification depth compared with conventional manual MAX enrichment or MWCO filtration alone, without introducing detectable glycan-type bias. Sensitivity analysis across a fourfold range of serum input volumes (5-20 μL) showed stable identification performance and quantitative reproducibility, with 10 μL identified as an optimal standardized input. Day-to-day reproducibility assessments further confirmed consistent quantitative performance across different acquisition densities. Application of the workflow to hepatocellular carcinoma (HCC) serum samples enabled reproducible, large-scale quantitative glycoproteomic profiling and revealed extensive disease-associated glycosylation remodeling, including site-specific regulation within individual proteins. Collectively, the 3kMAX platform provides a scalable solution for site-specific serum N-glycoproteomics and facilitates its translation into clinical and biomarker-oriented applications.

Keywords:  Automated glycopeptide enrichment; Depletion-free; High-throughput glycoproteomics; N-glycoproteomics; Serum; Site-specific glycosylation

DOI:  https://doi.org/10.1016/j.mcpro.2026.101611
Membranes (Basel). 2026 Jun 22. pii: 215. [Epub ahead of print]16(6):

Optimized Digestion Conditions for Membrane Protein Footprinting and Mass Spectrometry Analysis.

Ming Cheng, Xinzhu Li, Lin Bai, Weikai Li, Michael L Gross.

  Integral membrane proteins (IMPs), which constitute 50-60% of drug targets, play essential roles in numerous biological processes but remain underrepresented in conventional bottom-up and structural proteomics owing to their hydrophobicity and resistance to proteolysis. Although advances in IMP proteomics have improved global IMP detection, most efforts focus on proteome-scale protein identification rather than targeted structural analysis. Protein footprinting and cross-linking, two approaches in structural proteomics, require high sequence coverage and protein digestion to peptides of suitable length for structural elucidation, necessitating optimized digestion condition for individual IMPs. Here, we report a digestion protocol tailored for structural mass spectrometry and evaluate its performance by using a single amphipathic IMP model featuring distinct extramembrane and transmembrane domains. We evaluated the use of various protease-additive combinations and applied filter-aided sample preparation (FASP) to remove detergents and surfactants efficiently prior to MS analysis. The optimized conditions consistently yielded >90% sequence coverage. Guided by MS retention time calibration and hydrophobic factor simulations, we identified a "sweet spot" for transmembrane peptide detection. Notably, although cleavable surfactants can enhance proteome-wide coverage, our results show that they are not essential for single protein studies as they are in structural proteomics. Instead, detergent removal, protease selection, and generation of suitably sized peptides are critical for enabling reliable bottom-up structural analysis of IMPs. The protocol developed here provides a practical framework for optimizing digestion conditions in IMP characterization.

Keywords:  filter-aided sample preparation (FASP); mass spectrometry; membrane protein; membrane protein digestion; protein footprinting; vitamin K epoxide reductase (VKOR)

DOI:  https://doi.org/10.3390/membranes16060215