bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2025–02–23
twenty-one papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz
  1. A systematic analysis of in-source fragments in LC-MS metabolomics.
  2. From Complexity to Clarity: Expanding Metabolome Coverage With Innovative Analytical Strategies.
  3. Anaplerosis by medium-chain fatty acids through complex interplay with glucose and glutamine metabolism.
  4. SimMS: A GPU-Accelerated Cosine Similarity implementation for Tandem Mass Spectrometry.
  5. Recent advances in lipid analysis by capillary electromigration methods, 2019-2024.
  6. Phosphorylated glycosphingolipids are commonly detected in Caenorhabditis elegans lipidomes.
  7. Comprehensive tissue homogenization and metabolite extraction for application in clinical metabolomics.
  8. Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Identify Covalent TLCD1 Inhibitors.
  9. Structural Lipidomics Enabled by Isomer-Resolved Tandem Mass Spectrometry.
  10. An organism-level quantitative flux model of energy metabolism in mice.
  11. Swift Universal Glycan Acquisition (SUGA) Enables Quantitative Glycan Profiling across Diverse Sample Types.
  12. Sonication-assisted protein extraction improves proteomic detection of membrane-bound and DNA-binding proteins from tumor tissues.
  13. Direct lipid analysis of exosomes in serum by online miniaturized asymmetrical flow field-flow fractionation and electrospray ionization-mass spectrometry: Application to extrahepatic cholangiocarcinoma.
  14. From Reverse Phase Chromatography to HILIC: Graph Transformers Power Method-Independent Machine Learning of Retention Times.
  15. Isotope tracing-assisted chip-based solid-phase extraction mass spectrometry for monitoring metabolic changes and vitamin D3 regulation in cells.
  16. Reducing offsite modifications using 2-mercaptoethanol for LC-MS analyses.
  17. Mass spectrometry-based ligand binding assays in biomedical research.
  18. Discovery of Cysteine Carboxyalkylations by Real-Time Isotopic Signature Targeted Profiling.
  19. Critical overview of mass spectrometry-based lipidomics approach for evaluating lipid oxidation in foods.
  20. Evaluation of commercial micro-flow liquid chromatography columns for mass spectrometry-based proteomics.
  21. Plasma proteome variation and its genetic determinants in children and adolescents.
  1. bioRxiv. 2025 Feb 05. pii: 2025.02.04.636472. [Epub ahead of print]
    A systematic analysis of in-source fragments in LC-MS metabolomics.
    Yuanye Chi, Joshua M Mitchell, Shuzhao Li.
      The "dark matter" of metabolomics refers to the large number of unidentified features in metabolomic studies, mostly from mass spectrometry (MS) based analysis (deSilva2015; David2021; Giera2024). The topic is pertinent to the analytical coverage of small molecules in biomedical research (Kind2009; Uppal2016), approaches to metabolite annotation (Domingo2018; Chaleckis2019; Metz2025), mapping reaction pathways (Zamboni2015) and the promise of applying metabolomics and exposomics to precision medicine (Wishart2016; Vermeulen2020). The number of unidentified features is not a direct account of number of compounds, as a metabolite can have isotopologues, adducts and fragments that are measured in the same data (Mahieu2017; Wang2019; Li2023a). Giera et al (2024) recently reported that in-source fragments (ISFs) accounted for over 70% of MS/MS features in METLIN, one of the leading spectral databases, suggesting that ISFs could be a significant portion of the "dark matter". Since the reference spectra in METLIN are based on chemical standards, we examine here the LC-MS (liquid chromatography coupled mass spectrometry) metabolomics from biological samples, which are the most relevant in biomedical investigations.
    DOI:  https://doi.org/10.1101/2025.02.04.636472
  2. J Sep Sci. 2025 Feb;48(2): e70099
    From Complexity to Clarity: Expanding Metabolome Coverage With Innovative Analytical Strategies.
    Kanukolanu Aarika, Ramijinni Rajyalakshmi, Lakshmi Vineela Nalla, Siva Nageswara Rao Gajula.
      Metabolomics, a powerful discipline within systems biology, aims at comprehensive profiling of small molecules in biological samples. The challenges of biological sample complexity are addressed through innovative sample preparation methods, including solid-phase extraction and microextraction techniques, enhancing the detection and quantification of low-abundance metabolites. Advances in chromatographic separation, particularly liquid chromatography (LC) and gas chromatography (GC), coupled with high-resolution (HR) mass spectrometry (MS), have significantly improved the sensitivity, selectivity, and throughput of metabolomic studies. Cutting-edge techniques, such as ion-mobility mass spectrometry (IM-MS) and tandem MS (MS/MS), further expand the capacity for comprehensive metabolite profiling. These advanced analytical platforms each offer unique advantages for metabolomics, with continued technological improvements driving deeper insights into metabolic pathways and biomarker discovery. By providing a detailed overview of current trends and techniques, this review aims to offer valuable insights into the future of metabolomics in human health research and its translational potential in clinical settings. Toward the end, this review also highlights the biomedical applications of metabolomics, emphasizing its role in biomarker discovery, disease diagnostics, personalized medicine, and drug development.
    Keywords:  hydrophilic interaction liquid chromatography; ion‐mobility mass spectrometry; liquid chromatography–mass spectrometry (LC–MS); metabolomics; super critical fluid chromatography
    DOI:  https://doi.org/10.1002/jssc.70099
  3. J Biol Chem. 2025 Feb 13. pii: S0021-9258(25)00155-3. [Epub ahead of print] 108307
    Anaplerosis by medium-chain fatty acids through complex interplay with glucose and glutamine metabolism.
    Hannah M German, Jolita Ciapaite, Nanda M Verhoeven-Duif, Judith J M Jans.
      The constant replenishment of tricarboxylic acid (TCA) cycle intermediates, or anaplerosis, is crucial to ensure optimal TCA cycle activity in times of high biosynthetic demand. In inborn metabolic diseases, anaplerosis is often affected, leading to impaired TCA cycle flux and ATP production. In these cases, anaplerotic compounds can be a therapy option. Triheptanoin, a triglyceride containing three heptanoate chains, is thought to be anaplerotic through production of propionyl- and acetyl-CoA. However, the precise mechanism underlying its anaplerotic action remains poorly understood. In this study, we performed a comprehensive in vitro analysis of heptanoate metabolism and compared it to that of octanoate, an even-chain fatty acid which only provides acetyl-CoA. Using stable isotope tracing, we demonstrate that both heptanoate and octanoate contribute carbon to the TCA cycle in HEK293T cells, confirming direct anaplerosis. Furthermore, by using labeled glucose and glutamine, we show that heptanoate and octanoate decrease the contribution of glucose-derived carbon and increase the influx of glutamine-derived carbon into the TCA cycle. Our findings also point towards a change in redox homeostasis, indicated by an increased NAD+/NADH ratio, accompanied by a decreased lactate/pyruvate ratio and increased de novo serine biosynthesis. Taken together, these results highlight the broad metabolic effects of heptanoate and octanoate supplementation, suggesting that therapeutic efficacy may strongly depend on specific disease pathophysiology. Furthermore, they underline the need for careful selection of fatty acid compound and concentration to optimize anaplerotic action.
    Keywords:  Anaplerosis; fatty acids; isotopic tracer; mass spectrometry (MS); metabolic disease; metabolomics; redox regulation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108307
  4. Bioinformatics. 2025 Feb 20. pii: btaf081. [Epub ahead of print]
    SimMS: A GPU-Accelerated Cosine Similarity implementation for Tandem Mass Spectrometry.
    Tornike Onoprishvili, Jui-Hung Yuan, Kamen Petrov, Vijay Ingalalli, Lila Khederlarian, Niklas Leuchtenmuller, Sona Chandra, Aurelien Duarte, Andreas Bender, Yoann Gloaguen.
       MOTIVATION: Untargeted metabolomics involves a large-scale comparison of the fragmentation pattern of a mass spectrum against a database containing known spectra. Given the number of comparisons involved, this step can be time-consuming.
    RESULTS: In this work, we present a GPU-accelerated cosine similarity implementation for Tandem Mass Spectrometry (MS), with an approximately 1000-fold speedup compared to the MatchMS reference implementation, without any loss of accuracy. This improvement enables repository-scale spectral library matching for compound identification without the need for large compute clusters. This impact extends to any spectral comparison-based methods such as molecular networking approaches and analogue search.
    AVAILABILITY: All code, results, and notebooks supporting are freely available under the MIT license at https://github.com/pangeAI/simms/.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btaf081
  5. J Chromatogr A. 2025 Feb 12. pii: S0021-9673(25)00105-0. [Epub ahead of print]1746 465756
    Recent advances in lipid analysis by capillary electromigration methods, 2019-2024.
    Pierre Gavard, Amélie Gavard, Lucie Perquis, Fabrice Collin, François Couderc.
      Following a long period during which Capillary Electrophoresis (CE) was little used for lipid analysis (see Poinsot et al., Electrophoresis, 40, 2019, 190-211), the last five years have seen an increase in publications on this subject. Micellar Electrokinetic Chromatography (MEKC) can now compete with Gas Chromatography (GC) for the analysis of fatty acids, while non-aqueous capillary electrophoresis (NACE) now allows the study of fatty acids as well as phospholipids or glycolipids. As NACE also allows easy coupling to Mass Spectrometry (MS) in both positive and negative Electrospray Ionization (ESI), the technique has now become sufficiently robust, and for laboratories equipped with GC or Liquid Chromatography (LC) to consider using CE, particularly as it presents the advantage of much faster sample preparation than with GC for fatty acids and a resolution identical to LC for phospholipids and glycolipids. In this article, we will therefore describe the advances made in this area over the last five years.
    Keywords:  Fatty acids; Lipids; Micellar kinetic chromatography; Non-aqueous capillary electrophoresis; Phospholipids
    DOI:  https://doi.org/10.1016/j.chroma.2025.465756
  6. Metabolomics. 2025 Feb 20. 21(2): 29
    Phosphorylated glycosphingolipids are commonly detected in Caenorhabditis elegans lipidomes.
    Michael Witting, Liesa Salzer, Sven W Meyer, Aiko Barsch.
       INTRODUCTION: The identification of lipids is a cornerstone of lipidomics, and due to the specific characteristics of lipids, it requires dedicated analysis workflows. Identifying novel lipids and lipid species for which no reference spectra are available is tedious and often involves a lot of manual work. Integrating high-resolution mass spectrometry with enhancements from chromatographic and ion mobility separation enables the in-depth investigation of intact lipids.
    OBJECTIVES: We investigated phosphorylated glycosphingolipids from the nematode Caenorhabditis elegans, a biomedical model organism, and aimed to identify different species from this class of lipids, which have been described in one particular publication only. We checked if these lipids can be detected in lipid extracts of C. elegans.
    METHODS: We used UHPLC-UHR-TOF-MS and UHPLC-TIMS-TOF-MS in combination with dedicated data analysis to check for the presence of phosphorylated glycosphingolipids. Specifically, candidate features were identified in two datasets using Mass Spec Query Language (MassQL) to search fragmentation data. The additional use of retention time (RT) and collisional cross section (CCS) information allowed to filter false positive annotations.
    RESULTS: As a result, we detected all previously described phosphorylated glycosphingolipids and novel species as well as their biosynthetic precursors in two different lipidomics datasets. MassQL significantly speeds up the process by saving time that would otherwise be spent on manual data investigations. In total over 20 sphingolipids could be described.
    CONCLUSION: MassQL allowed us to search for phosphorylated glycosphingolipids and their potential biosynthetic precursors systematically. Using orthogonal information such as RT and CCS helped filter false positive results. With the detection in two different datasets, we demonstrate that these sphingolipids are a general part of the C. elegans lipidome.
    Keywords:   Caenorhabditis elegans ; Lipid identification; Lipidomics; MassQL; Sphingolipids; Trapped ion mobility
    DOI:  https://doi.org/10.1007/s11306-024-02216-w
  7. Anal Chim Acta. 2025 Mar 22. pii: S0003-2670(25)00122-9. [Epub ahead of print]1344 343728
    Comprehensive tissue homogenization and metabolite extraction for application in clinical metabolomics.
    Yuntao Hao, Jeannie Horak, Zorica Stijepic, Sultan Nilay Can, Luke Tu, Julia Alexandra Wolff, Berthold Koletzko.
       BACKGROUND: Metabolomics and lipidomics analysis of various biological samples offer insights into potential mechanisms of health and disease development. Tissue samples, compared to other biological samples, are less elucidated due to challenges in sample collection and lack of standardized sample preparation protocols for reproducible tissue homogenization and broad-range metabolite extraction.
    RESULTS: Pork tissue samples were homogenized with six different solvent mixtures with increasing lipophilicity, followed by metabolites extraction using methanol for polar and methyl-tert-butyl ether (MTBE) in methanol (MeOH) for highly lipophilic compounds. Metabolite profiles of supernatant and homogenate extraction for three extract volumes were compared. Solvent dependent pipette tip blockage was addressed by introduction of a prewetting correction factor for non-polar homogenization solutions and low volume tissue homogenate pipetting. Upset plots were applied for multi-dimensional metabolite extraction efficiency evaluation for 24 different sample preparation conditions. The best-performing homogenization solution was PBS; MeOH (1:1; v/v), combined with a two-step polar metabolite and lipid extraction using MeOH and 75 % MTBE in MeOH employing the tissue homogenate. The optimized experimental conditions were applied on mouse pancreas tissues, providing evidence of varying metabolic pathway activities across different anatomical regions of an organ.
    SIGNIFICANCE: This study introduces a comprehensive tissue sample preparation and metabolite quantification workflow, covering highly polar to highly lipophilic metabolites using targeted high performance liquid chromatography electrospray ionization triple quadrupole-linear ion trap mass spectrometer (HPLC-ESI-QTRAP-MS/MS) for absolute quantitation of amino acids, organic acids and keto-acids, acyl-carnitines, and phospho-choline lipids.
    Keywords:  HPLC-ESI-QTRAP-MS/MS; Homogenate; Lipidomics; Metabolomics; Prewetting; Tissue homogenization; Upset plot
    DOI:  https://doi.org/10.1016/j.aca.2025.343728
  8. bioRxiv. 2025 Jan 31. pii: 2025.01.31.635883. [Epub ahead of print]
    Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Identify Covalent TLCD1 Inhibitors.
    Hayden A Sharma, Michael Bielecki, Meredith A Holm, Ty M Thompson, Yue Yin, Jacob B Cravatt, Timothy B Ware, Alex Reed, Molham Nassir, Tamara El-Hayek Ewing, Bruno Melillo, J Fernando Bazan, Phil S Baran, Benjamin F Cravatt.
      Activity-based protein profiling (ABPP) of stereoisomerically defined sets of electrophilic compounds ('stereoprobes') offers a versatile way to discover covalent ligands for proteins in native biological systems. Here we report the synthesis and chemical proteomic characterization of stereoprobes bearing a P(V)-oxathiaphospholane (OTP) reactive group. ABPP experiments identified numerous proteins in human cancer cells that showed stereoselective reactivity with OTP stereoprobes, and we confirmed several of these liganding events with recombinant proteins. OTP stereoprobes engaging the poorly characterized transmembrane protein TLCD1 impaired the incorporation of monounsaturated fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic phenotype that mirrored genetic disruption of this protein. Using AlphaFold2, we found that TLCD1 structurally resembles the ceramide synthase and fatty acid elongase families of coenzyme A-dependent lipid processing enzymes. This structural similarity included conservation of catalytic histidine residues, the mutation of which blocked the OTP stereoprobe reactivity and lipid remodeling activity of recombinant TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid acyltransferase in cells, and that OTP stereoprobes function as inhibitors of this enzymatic activity. Our findings thus illuminate how the chemical proteomic analysis of electrophilic compounds can facilitate the functional annotation and chemical inhibition of a key lipid metabolic enzyme in human cells.
    DOI:  https://doi.org/10.1101/2025.01.31.635883
  9. Anal Chem. 2025 Feb 17.
    Structural Lipidomics Enabled by Isomer-Resolved Tandem Mass Spectrometry.
    Yikun Liu, Yu Xia, Wenpeng Zhang.
      
    DOI:  https://doi.org/10.1021/acs.analchem.4c06680
  10. Cell Metab. 2025 Feb 13. pii: S1550-4131(25)00008-7. [Epub ahead of print]
    An organism-level quantitative flux model of energy metabolism in mice.
    Bo Yuan, Will Doxsey, Özlem Tok, Young-Yon Kwon, Yanshan Liang, Karen E Inouye, Gökhan S Hotamışlıgil, Sheng Hui.
      Mammalian tissues feed on nutrients in the blood circulation. At the organism level, mammalian energy metabolism is comprised of the oxidation, storage, interconversion, and release of circulating nutrients. Here, by integrating isotope tracer infusion, mass spectrometry, and isotope gas analyzer measurement, we developed a framework to systematically quantify fluxes through these metabolic processes for 10 major circulating energy nutrients in mice, resulting in an organism-level quantitative flux model of energy metabolism. This model revealed in wild-type mice that circulating nutrients have metabolic cycling fluxes dominant to their oxidation fluxes, with distinct partitions between cycling and oxidation for individual circulating nutrients. Applications of this framework in obese mouse models showed extensive elevation of metabolic cycling fluxes in ob/ob mice but not in diet-induced obese mice on a per-animal or per-lean mass basis. Our framework is a valuable tool to reveal new features of energy metabolism in physiological and disease conditions.
    Keywords:  energy metabolism; futile cycle; high-fat diet; isotope tracing; metabolic flux analysis; ob/ob; obesity
    DOI:  https://doi.org/10.1016/j.cmet.2025.01.008
  11. J Proteome Res. 2025 Feb 20.
    Swift Universal Glycan Acquisition (SUGA) Enables Quantitative Glycan Profiling across Diverse Sample Types.
    Christopher Ashwood, Cecilia Voelcker, Richard D Cummings.
      The ability to rapidly analyze complex mixtures of glycans derived from glycoproteins is important, but techniques are often laborious and require multiple glycan derivatization steps. Here, we describe an approach termed Swift Universal Glycan Acquisition (SUGA) in which the total released, nonreduced N-glycan samples are analyzed following direct injection and electrospray ionization in a mass spectrometer with a rapid 3 min run time for each sample. As electrospray ionization (ESI) can generate multiple charge states and adducts for the same glycan composition (MS1), deconvolution is performed to yield the relative intensity profile for each detected glycan composition; each annotated composition is supported by an annotated MS2 spectrum. This combination of MS1 and MS2 data enables confident glycan identification. The data obtained by SUGA are comparable to those obtained using permethylated N-glycans analyzed by matrix-assisted laser desorption/ionization (MALDI)-MS. The SUGA approach was applied to the analyses of several purified glycoproteins and N-glycans derived from cells and compared to spectra obtained following permethylation and analysis by MALDI-MS. This new approach will facilitate the rapid and high-throughput analysis of N-glycans from diverse biological samples.
    Keywords:  SUGA; glycomics; high-throughput; inhibitor screening; time-course
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00657
  12. Nat Protoc. 2025 Feb 17.
    Sonication-assisted protein extraction improves proteomic detection of membrane-bound and DNA-binding proteins from tumor tissues.
    Qing Kay Li, T Mamie Lih, David J Clark, Lijun Chen, Michael Schnaubelt, Hui Zhang.
      Deep-scale, mass spectrometry-based proteomic studies by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) program involves tissue lysis using urea buffer before data acquisition via mass spectrometry for quantitative global proteomic and phosphoproteomic analysis. This is described in a 2018 protocol1. Here we report an update to this initial protocol by implementing a sonication step into urea-based tissue lysis. Similar to the initial CPTAC protocol, we identified >12,000 proteins and >25,000 phosphopeptides in a tandem mass tag (TMT) set containing both nonsonicated and sonicated tumor tissues from patient-derived xenograft mouse models. An improvement in the detection of membrane-bound and DNA-binding proteins was observed by including the sonication. We also offer recommendations for optimal sonication conditions such as the buffer composition, timing of sonication cycle, instrumentation settings and a troubleshooting section for potential users. Additionally, the protocol is equally applicable to other biological specimens.
    DOI:  https://doi.org/10.1038/s41596-024-01113-9
  13. J Chromatogr A. 2025 Feb 14. pii: S0021-9673(25)00126-8. [Epub ahead of print]1746 465778
    Direct lipid analysis of exosomes in serum by online miniaturized asymmetrical flow field-flow fractionation and electrospray ionization-mass spectrometry: Application to extrahepatic cholangiocarcinoma.
    Hye Ju Yu, Myeong Hee Moon.
      Exosomes are submicron-sized extracellular vesicles involved in immune regulation, tumor metastasis, and cellular communication. Their lipid composition, distinct from parental cells, plays a crucial role in diseases like cancer. However, lipidomic analysis of exosomes, particularly in complex samples like blood, requires advanced techniques. This study optimizes miniaturized flow field-flow fractionation (mFlFFF) coupled with electrospray ionization mass spectrometry (ESI-MS) for direct lipidomic analysis of exosomes in serum. The mFlFFF technique resolves exosomes for size-based lipid analysis without prior extraction. Lipidomic profiling of serum exosomes from patients with extrahepatic cholangiocarcinoma (eCCA) identified over 1000 lipid species, with 64 showing significant changes compared to healthy controls. Target lipids were analyzed by mFlFFF-ESI-MS, revealing 35 species that distinguish eCCA patients from controls, suggesting their potential as biomarkers. Elevated levels of lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (PI) were observed in the eCCA group, indicating lipid alterations linked to cancer progression and inflammation. Notably, PI 38:4, involved in the release of arachidonic acid, highlights its role in inflammatory processes associated with cancer. This study demonstrates the potential of mFlFFF-ESI-MS for lipidomic analysis of exosomes and offers a non-invasive approach for cancer diagnosis, with future implications for therapeutic targeting of lipid pathways in cholangiocarcinoma.
    Keywords:  Exosome; Extrahepatic cholangiocarcinoma; Flow FFF; Lipidomics; Mass spectrometry; mFlFFF-ESI-MS
    DOI:  https://doi.org/10.1016/j.chroma.2025.465778
  14. Anal Chem. 2025 Feb 19.
    From Reverse Phase Chromatography to HILIC: Graph Transformers Power Method-Independent Machine Learning of Retention Times.
    Cailum M K Stienstra, Emir Nazdrajić, W Scott Hopkins.
      Liquid chromatography (LC) is a cornerstone of analytical separations, but comparing the retention times (RTs) across different LC methods is challenging because of variations in experimental parameters such as column type and solvent gradient. Nevertheless, RTs are powerful metrics in tandem mass spectrometry (MS2) that can reduce false positive rates for metabolite annotation, differentiate isobaric species, and improve peptide identification. Here, we present Graphormer-RT, a novel graph transformer that performs the first single-model method-independent prediction of RTs. We use the RepoRT data set, which contains 142,688 reverse phase (RP) RTs (from 191 methods) and 4,373 HILIC RTs (from 49 methods). Our best RP model (trained and tested on 191 methods) achieved a test set mean average error (MAE) of 29.3 ± 0.6 s, comparable performance to the state-of-the-art model which was only trained on a single LC method. Our best-performing HILIC model achieved a test MAE = 42.4 ± 2.9 s. We expect that Graphormer-RT can be used as an LC "foundation model", where transfer learning can reduce the amount of training data needed for highly accurate "specialist" models applied to method-specific RP and HILIC tasks. These frameworks could enable the machine optimization of automated LC workflows, improved filtration of candidate structures using predicted RTs, and the in silico annotation of unknown analytes in LC-MS2 measurements.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05859
  15. Talanta. 2025 Feb 15. pii: S0039-9140(25)00244-9. [Epub ahead of print]288 127754
    Isotope tracing-assisted chip-based solid-phase extraction mass spectrometry for monitoring metabolic changes and vitamin D3 regulation in cells.
    Ning Xu, Haifeng Lin, Xiaodan Ding, Peilong Wang, Jin-Ming Lin.
      Cellular metabolism is a dynamic and essential process, with alterations in metabolic pathways serving as hallmark features of cancer. In this study, we developed a chip-based solid-phase extraction mass spectrometry (Chip-SPE-MS) platform for high-sensitivity, high-throughput analysis of cellular metabolites and real-time tracking of metabolic fluxes. The system achieved detection limits ranging from 0.10 to 9.43 μmol/mL for various amino acids and organic acids, with excellent linearity (r ≥ 0.992). By incorporating isotope tracing, the platform enabled derivatization-free, real-time monitoring of 13C-labeled metabolites, such as lactic acid. Our analysis revealed significant metabolic differences between normal (L02) and cancerous (HepG2, HCT116) cells, including enhanced glycolytic activity and elevated lactate production in cancer cells. Furthermore, treatment with 1,25-dihydroxyvitamin D3 was shown to suppress glucose uptake and modulate metabolic activity in HCT116 cells, highlighting the regulatory effects of vitamin D3 on cancer metabolism. This study not only provides novel insights into the metabolic reprogramming associated with cancer but also demonstrates the potential of the Chip-SPE-MS platform as a powerful tool for real-time monitoring of dynamic metabolic processes. The findings have broad implications for cancer therapy and the study of metabolic diseases.
    Keywords:  Mass spectrometry; Metabolic change; Microfluidics chip; Vitamin D3
    DOI:  https://doi.org/10.1016/j.talanta.2025.127754
  16. Biochem Biophys Res Commun. 2025 Feb 12. pii: S0006-291X(25)00199-8. [Epub ahead of print]753 151485
    Reducing offsite modifications using 2-mercaptoethanol for LC-MS analyses.
    Arisa Suto, Takashi Matsui, Yoshio Kodera.
      The alkylation of thiol groups in cysteine (Cys) residues is a critical step in liquid chromatography-mass spectrometry (LC-MS) analysis. However, conventional alkylation methods often introduce non-specific modifications at the N-terminal and other amino acids, thus complicating peptide identification and quantification using MS. To overcome this problem, we developed a novel method using 2-mercaptoethanol (2-ME) in the presence of dimethyl sulfoxide (DMSO) to specifically target Cys residues. Although 2-ME is typically used as a reducing agent for disulfide bonds, we demonstrated that DMSO promotes the specific binding of 2-ME to Cys in a concentration-dependent manner. Importantly, this approach significantly reduces offsite alkylation of other amino acids compared with conventional procedures, thereby improving peptide identification and enhancing the overall accuracy of quantification. This method offers a practical solution to the challenges posed by conventional alkylation techniques, provides more reliable MS data for peptides containing Cys residues, and enhances the accuracy of peptide quantification.
    Keywords:  2-Mercaptoethanol; Cysteine modification; Dimethyl sulfoxide
    DOI:  https://doi.org/10.1016/j.bbrc.2025.151485
  17. Expert Rev Proteomics. 2025 Feb 18.
    Mass spectrometry-based ligand binding assays in biomedical research.
    Tobias Meisinger, Amelie Vogt, Robin Kretz, Helen S Hammer, Hannes Planatscher, Oliver Poetz.
       INTRODUCTION: Ligand binding assays combining immunoaffinity enrichment steps with mass spectrometry (MS) readout have gained attention as a highly specific and sensitive tool for protein quantification. These techniques typically combine enzymatic fragmentation of the sample or enriched protein with capture on the protein or peptide-level for quantification. Antibodies ensure specific target recognition, while MS offers quantitative accuracy with isotopically labeled internal standards. This dual approach supports a broad dynamic range, enabling protein measurements from picomolar to nanomolar levels. These methods have diverse applications, from quantifying signaling proteins in basic research to biomarker monitoring in clinical trials and analyzing the pharmacokinetics of therapeutic proteins.
    AREAS COVERED: This review delves into the diverse workflows of immunoaffinity-MS, shedding light on the innovative strategies employed, their practical applications, efficacy, and inherent limitations in the realm of protein quantification.
    EXPERT OPINION: Immunoaffinity-MS has transformed protein analysis, but widespread adoption is hindered by complex workflows, high instrument costs, and limited capture molecule availability. Efforts to enhance automation, standardize workflows, and advance technological innovation aim to overcome these barriers. Improvements in mass spectrometer sensitivity, advances in recombinant capture technologies, and support from public initiatives are poised to further improve the reliability and accessibility of this method.
    Keywords:  Biomarker; Immuno-MRM; TXP antibodies; iMALDI; immunoaffinity LC-MS/MS; mass spectrometry
    DOI:  https://doi.org/10.1080/14789450.2025.2467263
  18. J Am Chem Soc. 2025 Feb 21.
    Discovery of Cysteine Carboxyalkylations by Real-Time Isotopic Signature Targeted Profiling.
    Guogeng Jia, Yicheng Liu, Tianyu Feng, Zixuan Zhang, Huan Tang, Fan Yang, Ziyao Tang, Weidi Xiao, Ying Chen, Jinjun Gao, Chu Wang.
      Data-dependent acquisition (DDA) is widely applied in shotgun proteomics. However, restricted by the scanning speed of mass spectrometry (MS) instruments, it remains challenging for DDA to directly detect peptides with low abundance. Herein, we developed a real-time targeted MS data acquisition method, "isoSTAR", which identifies target peptides by their unique isotopic signatures during the stage of full-MS scanning and subjects them to targeted MS/MS scans immediately. The method showed dramatic improvement in sensitivity in identifying target peptides with low abundance compared to traditional MS acquisition methods. Using this method, we discovered a series of carboxyalkylations on cysteines during fatty acid metabolism and verified their modification structures using synthetic peptide standards. We envision that isoSTAR will become a powerful and versatile tool to enhance shotgun proteomics applications in profiling protein-centric modifications.
    DOI:  https://doi.org/10.1021/jacs.4c16183
  19. Food Sci Biotechnol. 2025 Mar;34(4): 837-849
    Critical overview of mass spectrometry-based lipidomics approach for evaluating lipid oxidation in foods.
    JaeYoon Kang, JuDong Yeo.
      Mass spectrometry-based lipidomics, developed through rapid advancements in instruments and techniques, provides comprehensive analyses of individual lipidomes in diverse biological systems. This contribution summarizes the limitations of classical methods for measuring lipid oxidation in foods and presents current novel technologies for evaluating lipid oxidation. Notably, this study introduces the mass spectrometry-based lipidomics approach and its utility in assessing lipid oxidation through various analytical modes, supported by numerous examples. This overview offers significant insights into the use of mass spectrometry-based lipidomics for measuring lipid oxidation in foods, proposing lipidomics analysis as a promising method to address the limitations of classical approaches.
    Keywords:  LC–MS/MS; Lipid oxidation; Lipidomics
    DOI:  https://doi.org/10.1007/s10068-024-01726-6
  20. J Chromatogr A. 2025 Feb 18. pii: S0021-9673(25)00137-2. [Epub ahead of print]1746 465789
    Evaluation of commercial micro-flow liquid chromatography columns for mass spectrometry-based proteomics.
    Haiyue Zuo, Dandan Zhou, Chunli Gao, Yang Yang, Lin Cao, Sha Liao, Junjie Ou, Yangyang Bian.
      This study aimed to evaluate the separation efficiency and loading capacity of four commercially available micro-flow liquid chromatography (micro-flow LC) columns with 1.0 mm i.d. (PepMap™ C18, HALO® ES-C18, YMC-Triart™ C18 and Acquity UPLC Peptide BEH C18, abbreviated as PepMap, HALO, YMC and BEH) for mass spectrometry-based proteomics analysis. Samples including cytochrome c (cyt-c), human plasma, and HeLa protein digest were used for the tests. The YMC showed much wider peak widths than the other columns, exhibited relatively poor identification results. However, the other three columns showed similar identification performance. Among them, the PepMap was the optimal choice for plasma proteomics as it had a high loading capacity and exhibited the most symmetrical peak shape with a symmetry factor closest to 1.0. In general, our results provided valuable and solid support for the choice of a chromatographic column, which could potentially contributes to the wider application of micro-flow LC-MS/MS in proteomics research.
    Keywords:  Column evaluation; Micro-flow LC-MS/MS; Peptide identification; Proteomics
    DOI:  https://doi.org/10.1016/j.chroma.2025.465789
  21. Nat Genet. 2025 Feb 19.
    Plasma proteome variation and its genetic determinants in children and adolescents.
    Lili Niu, Sara Elizabeth Stinson, Louise Aas Holm, Morten Asp Vonsild Lund, Cilius Esmann Fonvig, Leonardo Cobuccio, Jonas Meisner, Helene Bæk Juel, Joao Fadista, Maja Thiele, Aleksander Krag, Jens-Christian Holm, Simon Rasmussen, Torben Hansen, Matthias Mann.
      Our current understanding of the determinants of plasma proteome variation during pediatric development remains incomplete. Here, we show that genetic variants, age, sex and body mass index significantly influence this variation. Using a streamlined and highly quantitative mass spectrometry-based proteomics workflow, we analyzed plasma from 2,147 children and adolescents, identifying 1,216 proteins after quality control. Notably, the levels of 70% of these were associated with at least one of the aforementioned factors, with protein levels also being predictive. Quantitative trait loci (QTLs) regulated at least one-third of the proteins; between a few percent and up to 30-fold. Together with excellent replication in an additional 1,000 children and 558 adults, this reveals substantial genetic effects on plasma protein levels, persisting from childhood into adulthood. Through Mendelian randomization and colocalization analyses, we identified 41 causal genes for 33 cardiometabolic traits, emphasizing the value of protein QTLs in drug target identification and disease understanding.
    DOI:  https://doi.org/10.1038/s41588-025-02089-2
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