bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2025–08–31
sixteen papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz



  1. J Vis Exp. 2025 Aug 08.
      Endogenous neuropeptides are key modulators of brain function, playing critical roles in behavior, stress, pain, and homeostatic regulation, yet their analysis remains difficult. Biologically, they are low in abundance, rapidly degraded, and processed variably from precursor proteins, with expression limited to small, localized cell populations. Technically, their detection is complicated by a wide dynamic range, diverse post-translational modifications, and sparse signals in mass spectrometry datasets. This protocol outlines a comprehensive workflow for neuropeptide analysis in Rattus norvegicus brain tissue using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry (MS) on a timsTOF platform. Following optimized brain sample preparation, including dissection, peptide extraction and clean-up, nano liquid chromatography (LC)-MS is performed with ion mobility gas-phase fractionation to improve detection sensitivity and accuracy. The DDA-generated spectral library supports DIA-based quantification in Skyline, enabling high-confidence MS2-level measurements. This integrated workflow increases neuropeptide coverage and enhances quantitative reproducibility, providing a robust platform for studying neuropeptides in complex brain tissue.
    DOI:  https://doi.org/10.3791/68741
  2. Nat Protoc. 2025 Aug 22.
      The direct coupling of ion-exchange chromatography with mass spectrometry using electrochemical ion suppression creates a hyphenated technique with selectivity and specificity for the analysis of highly polar and ionic compounds. The technique has enabled new applications in environmental chemistry, food chemistry, forensics, cell biology and, more recently, metabolomics. Robust, reproducible and quantitative methods for the analysis of highly polar and ionic metabolites help meet a longstanding analytical need in metabolomics. Here, we provide step-by-step instructions for both untargeted and semi-targeted metabolite analysis from cell, tissue or biofluid samples by using anion-exchange chromatography-high-resolution tandem mass spectrometry (AEC-MS/MS). The method requires minimal sample preparation and is robust, sensitive and selective. It provides comprehensive coverage of hundreds of metabolites found in primary and secondary metabolic pathways, including glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, purine and pyrimidine metabolism, amino acid degradation and redox metabolism. An inline electrolytic ion suppressor is used to quantitatively neutralize OH- ions in the eluent stream, after chromatographic separation, enabling AEC to be directly coupled with MS. Counter ions are also removed during this process, creating a neutral pH, aqueous eluent with a simplified matrix optimal for negative ion MS analysis. Sample preparation through to data analysis and interpretation is described in the protocol, including a guide to which metabolites and metabolic pathways are suitable for analysis by using AEC-MS/MS.
    DOI:  https://doi.org/10.1038/s41596-025-01222-z
  3. Metabolites. 2025 Jul 23. pii: 496. [Epub ahead of print]15(8):
       BACKGROUND: Liquid chromatography-mass spectrometry (LC-MS), widely used in metabolomics, is often limited by low ionization efficiency and ion suppression, which reduce overall metabolite detectability and quantification accuracy. To address these challenges, chemical isotope labeling (CIL) LC-MS has emerged as a powerful approach, offering high sensitivity, accurate quantification, and broad metabolome coverage. This method enables comprehensive profiling by targeting multiple submetabolomes. Specifically, amine-/phenol- and hydroxyl-containing metabolites are labeled using dansyl chloride under distinct reaction conditions. While this strategy provides extensive coverage, the sequential analysis of each submetabolome reduces throughput. To overcome this limitation, we propose a two-channel mixing strategy to improve analytical efficiency.
    METHODS: In this approach, samples labeled separately for the amine/phenol and hydroxyl submetabolomes are combined prior to LC-MS analysis, leveraging the common use of dansyl chloride as the labeling reagent. This integration effectively doubles throughput by reducing LC-MS runtime and associated costs. The method was evaluated using human urine and serum samples, focusing on peak pair detectability and metabolite identification. A proof-of-concept study was also conducted to assess the approach's applicability in putative biomarker discovery.
    RESULTS: Results demonstrate that the two-channel mixing strategy enhances throughput while maintaining analytical robustness.
    CONCLUSIONS: This method is particularly suitable for large-scale studies that require rapid sample processing, where high efficiency is essential.
    Keywords:  chemical isotope labeling (CIL) LC-MS; comprehensive metabolome profiling; dansyl labeling; rapid sample processing
    DOI:  https://doi.org/10.3390/metabo15080496
  4. Angew Chem Int Ed Engl. 2025 Aug 23. e202510692
      Single-cell mass spectrometry (MS) offers unprecedented sensitivity for profiling cellular proteomes, yet widespread adoption is hindered by the cost of advanced instrumentation. Here, we broaden access to single-cell proteomics by combining capillary electrophoresis (CE), data-dependent acquisition (DDA) with electrophoresis-correlative (Eco) ion sorting, and artificial intelligence (AI)-assisted spectral deconvolution via CHIMERYS (Eco-AI). This "Real-Time Eco-AI" workflow was implemented on a custom-built CE platform coupled to a legacy hybrid quadrupole-orbitrap mass spectrometer (Q Exactive Plus). Despite slower scan speed, lower resolution, and inferior ion transmission efficiency, real-time Eco-DDA sampling and CHIMERYS processing enabled identification of up to ∼15 peptides per spectrum-performance on par with modern Orbitrap Fusion Lumos tribrid systems. From 1 ng of HeLa digest, 2142 proteins were identified, surpassing the 969 proteins detected on a contemporary nanoLC Orbitrap Fusion Lumos. Even from ∼250 pg (a single-cell equivalent), 1799 proteins were identified in <15 min of effective separation, raising a theoretical throughput of 48 samples per day. As proof of principle, Real-Time Eco-AI profiled 1524 proteins from single precursor cells (50-75 µm diameter) in Xenopus laevis blastulae, revealing proteome asymmetry during neural versus epidermal fate specification. These results establish Real-Time Eco-AI as a budget-conscious yet powerful strategy for single-cell proteomics using CE-MS.
    Keywords:  Capillary electrophoresis; Intelligent data acquisition; Mass spectrometry; Proteomics; Single cell
    DOI:  https://doi.org/10.1002/anie.202510692
  5. Anal Chem. 2025 Aug 27.
      Mass spectrometry imaging (MSI) is a powerful tool for monitoring the spatial distributions of microbial metabolites directly from culture. MSI can identify secretion and retention patterns for microbial metabolites, allowing for the assessment of chemical communication within complex microbial communities. Microbial imaging via matrix-assisted laser desorption/ionization (MALDI) MSI remains challenging due to high sample complexity and heterogeneity associated with the required sample preparation, making annotation of molecules by MS1 alone challenging. The implementation of trapped ion mobility spectrometry (TIMS) has increased the dimensionality of MALDI-MSI experiments, allowing for the resolution of isomers and isobars, and can increase sensitivity of metabolite detection within complex samples. Parallel reaction monitoring-parallel accumulation serial fragmentation (prm-PASEF) leverages TIMS to enhance the targeted acquisition of MS2 data by increasing the number of precursors that can be fragmented in a single acquisition. Recently, imaging prm-PASEF (iprm-PASEF) has been developed to provide more accurate annotation from MALDI-TIMS-MSI data sets through the inclusion of MS2. Here, we showcase the use of MALDI iprm-PASEF to provide rapid and accurate annotation of coproporphyrin III directly from a bacterial-fungal coculture between Glutamicibacter arilaitensis (strain JB182) and Penicillium solitum (strain #12). Additionally, we present a workflow for untargeted iprm-PASEF precursor selection directly in SCiLS Lab, followed by direct export for iprm-PASEF acquisition.
    DOI:  https://doi.org/10.1021/acs.analchem.5c02787
  6. Anal Chem. 2025 Aug 26.
      The ability to answer complex biological questions in metabolomics relies on the acquisition of high-quality data. However, due to the complex nature of liquid chromatography-mass spectrometry acquisition, data quality checks are often not done comprehensively and only at the postprocessing step. This can be too late to mitigate analytical problems such as loss of m/z calibration, retention time drift and severe ion suppression. It is often not practically or economically feasible to reanalyze samples, and interpretation of the acquired compromised data, if at all possible, is limited, despite the considerable expenses incurred to obtain them. We therefore introduce QC4Metabolomics, a real-time quality control monitoring software for untargeted metabolomics data. QC4Metabolomics monitors files as they are acquired or retrospectively by tracking any user-defined compound(s) and extracting diagnostic information such as observed m/z, retention time, intensity and peak shape, and presents the results on a web dashboard. QC4Metabolomics also monitors the levels of common or user-defined contaminants. We report herein real-world examples where QC4Metabolomics easily reveals analytical problems retrospectively that could have been immediately addressed with real-time monitoring, so that the samples would have been analyzed without any quality control issues. The Shiny app is available as open-source code at https://github.com/stanstrup/QC4Metabolomics. Docker images and a docker-compose setup file are also provided for easy deployment, along with demo data. The documentation can be found at https://stanstrup.github.io/QC4Metabolomics.
    DOI:  https://doi.org/10.1021/acs.analchem.4c07078
  7. Metabolites. 2025 Jul 31. pii: 513. [Epub ahead of print]15(8):
      Mitochondria, pivotal organelles in cellular metabolism and energy production, have emerged as critical players in the pathogenesis of cancer. This review outlines the progress in mitochondrial profiling through mass spectrometry-based metabolomics and its applications in cancer research. We provide unprecedented insights into the mitochondrial metabolic rewiring that fuels tumorigenesis, metastasis, and therapeutic resistance. The purpose of this review is to provide a comprehensive guide for the implementation of mitochondrial metabolomics, integrating advanced methodologies-including isolation, detection, and data integration-with insights into cancer-specific metabolic rewiring. We first summarize current methodologies for mitochondrial sample collection and pretreatment. Furthermore, we then discuss the recent advancements in mass spectrometry-based methodologies that facilitate the detailed profiling of mitochondrial metabolites, unveiling significant metabolic reprogramming associated with tumorigenesis. We emphasize how recent technological advancements have addressed longstanding challenges in the field and explore the role of mitochondrial metabolism-driven cancer development and progression for novel drug discovery and translational research applications in cancer. Collectively, this review delineates emerging opportunities for therapeutic discovery and aims to establish a foundation for future investigations into the therapeutic modulation of mitochondrial pathways in cancer, thereby paving the way for innovative diagnostic and therapeutic approaches targeting mitochondrial pathways.
    Keywords:  mass spectrometry; mitochondria isolation; mitochondrial metabolomics; targeted metabolomics; untargeted metabolomics
    DOI:  https://doi.org/10.3390/metabo15080513
  8. Nat Commun. 2025 Aug 26. 16(1): 7934
      Organs collaborate to maintain metabolic homeostasis in mammals. Spatial metabolomics makes strides in profiling the metabolic landscape, yet can not directly inspect the metabolic crosstalk between tissues. Here, we introduce an approach to comprehensively trace the metabolic fate of 13C-nutrients within the body and present a robust computational tool, MSITracer, to deep-probe metabolic activity in a spatial manner. By discerning spatial distribution differences between isotopically labeled metabolites from ambient mass spectrometry imaging-based isotope tracing data, this approach empowers us to characterize fatty acid metabolic crosstalk between the liver and heart, as well as glutamine metabolic exchange across the kidney, liver, and brain. Moreover, we disclose that tumor burden significantly influences the host's hexosamine biosynthesis pathway, and that the glucose-derived glutamine released from the lung as a potential source for tumor glutamate synthesis. The developed approach facilitates the systematic characterization of metabolic activity in situ and the interpretation of tissue metabolic communications in living organisms.
    DOI:  https://doi.org/10.1038/s41467-025-63243-2
  9. J Proteome Res. 2025 Aug 20.
      In addition to their roles in energetics and biosynthesis, endogenous metabolites have functional roles performed in part through protein interactions that result in allosteric regulation, transcriptional regulation, and post-translational modifications. Novel bioactive roles for metabolites continue to emerge in cancer progression, immune response, and host-pathogen interactions. Defining metabolite-protein interactions will further reveal bioactive metabolite downstream effects and help to characterize the intersection between the metabolome and the proteome. Here, we summarize recently revealed secondary functions for metabolites that have been uncovered by mass spectrometry-based approaches for small molecule target engagement. We propose that further developments and application of these approaches will greatly advance our understanding of metabolite functions and may facilitate large-scale metabolome-proteome interaction networks that harbor new targets for diseases such as cancer.
    Keywords:  mass spectrometry; metabolite-protein interactions; metabolome; proteome; target engagement techniques
    DOI:  https://doi.org/10.1021/acs.jproteome.5c00341
  10. Crit Rev Oncol Hematol. 2025 Aug 18. pii: S1040-8428(25)00294-X. [Epub ahead of print]215 104906
      Cysteine metabolism plays a pivotal role in ferroptosis regulation by modulating antioxidant defense, lipid peroxidation, and iron homeostasis. Cancer cells exploit cysteine availability to evade ferroptotic cell death, contributing to tumor progression and therapy resistance. Despite growing interest in ferroptosis as a therapeutic vulnerability, a comprehensive understanding of cysteine metabolism in this process remains essential. This review explores key sources of intracellular cysteine, its roles in ferroptosis suppression, and therapeutic strategies targeting cysteine metabolism in cancer. We discuss systemic cysteine depletion, xCT inhibition, suppression of H2S biosynthesis, and GPX4-targeted therapies, along with promising drug combinations. While preclinical studies highlight the efficacy of these approaches, in vivo validation and clinical translation remain limited. Advancing cysteine-targeting therapies require further mechanistic insights, biomarker identification, and optimized delivery strategies. A deeper understanding of cysteine metabolism may pave the way for ferroptosis-based cancer treatments with improved precision and efficacy.
    Keywords:  Cancer therapy; Cyst(e)inases; Cysteine metabolism; Ferroptosis; XCT targeting
    DOI:  https://doi.org/10.1016/j.critrevonc.2025.104906
  11. J Proteome Res. 2025 Aug 26.
      Serum proteomics plays a crucial role in biomarker discovery and disease research, yet the selection of an optimal sample preparation method remains challenging. Evaluating the accuracy of protein quantitation is of major importance and a vital part of a benchmarking study in proteomics, since clinical studies are based on the differential expression of certain proteins that can be used as biomarkers or be indicative of a pathological state. In this study, we performed a direct comparison of 6 widely used serum proteomic sample preparation workflows: In-gel digestion (IGD), Single-Pot Solid-Phase-enhanced Sample Preparation (SP3), Top 14 Abundant Protein Depletion (Top 14), Isopropanol/Trichloroacetic Acid (IPA/TCA) precipitation, PreOmics ENRICH-iST (PreOmics), and Seer Proteograph XT (Seer). Seer and PreOmics demonstrated superior quantitative accuracy, especially for proteins with low abundance in serum, while the Seer enrichment approach provided the highest number of protein identifications (>2000) as measured by an Orbitrap Exploris 480. All methods had median CVs close to or below 20%. This comparative analysis provides a comprehensive resource for selecting the most appropriate serum sample preparation strategy based on specific experimental needs, facilitating human serum proteomic profiling for biomedical research.
    Keywords:  depth of proteome; quantitative accuracy; reproducibility; sample preparation methods; serum proteomics; shotgun analysis
    DOI:  https://doi.org/10.1021/acs.jproteome.5c00572
  12. Trends Biotechnol. 2025 Aug 22. pii: S0167-7799(25)00311-7. [Epub ahead of print]
      Residual host cell proteins (HCPs) in biologic drug products can compromise safety or stability and must be carefully monitored. While traditional immunoassays remain essential, they often lack specificity or coverage. Mass spectrometry (MS) offers a complementary approach by enabling direct identification and quantification of individual HCPs throughout development. This review highlights recent advances in MS technologies and workflows relevant to HCP detection, including new data acquisition strategies, software tools, and artificial intelligence applications. We also discuss regulatory perspectives and considerations for implementing MS in controlled environments. By integrating analytical innovations with risk-based strategies, MS-based approaches are becoming key components of modern biopharmaceutical quality control.
    Keywords:  artificial intelligence in proteomics; biopharmaceutical impurities; host cell proteins; mass spectrometry; regulatory analytics
    DOI:  https://doi.org/10.1016/j.tibtech.2025.07.026
  13. Anal Chem. 2025 Aug 26.
      Traditional lipidomics methods are not well-suited for studying unsaturated lipids containing located C═C bonds in single cells. Here, we proposed a single-cell mass spectrometry (MS) platform assisted by a photochemical derivatization (PCD) method termed Visible-light-activated Ru(bpy)32+-catalyzed Singlet Oxygen-mediated Hydroperoxidation (VRSOH), for the rapid and simultaneous detection of multiple lipid C═C location isomers in living cell membranes at the single-cell level. First, the VRSOH method combined with MS analysis achieved the localization of C═C bonds in mono- and polyunsaturated lipids and cellular lipid extracts. Then, based on the home-built rapid single-cell MS platform and Ru-L, a modified form of Ru(bpy)32+ that allowed the VRSOH method to be applied to living cell membranes, 15 lipid C═C location isomers from the top-ranked phosphatidylcholines (PCs) in both primary and metastatic colorectal cancer (CRC) cells were quantified with a throughput of ∼16 cells/min. The results also revealed specific lipidomic patterns associated with CRC metastasis and the existence of subgroups with distinct migration potentials in metastatic CRC cells. In light of the lipidomic pattern discovery, stearoyl-CoA desaturase 1 (SCD1) may serve as a potential target for CRC metastasis prevention.
    DOI:  https://doi.org/10.1021/acs.analchem.5c01725
  14. Nat Commun. 2025 Aug 20. 16(1): 7773
      Molecular glue degraders (MGDs) are small molecules that co-opt the ubiquitin-proteasome system to induce degradation of target proteins, including those considered undruggable. Their discovery remains challenging due to the lack of rational design strategies and limited throughput of unbiased proteome-wide screening approaches. To address this gap, we develop a high-throughput proteomics platform based on label-free, data-independent acquisition mass spectrometry (DIA-MS), enabling integrated proteomics and ubiquitinomics profiling. Screening a diverse set of 100 cereblon (CRBN)-recruiting ligands on this platform leads to identification of a broad array of novel degraders and neosubstrates. Subsequent hit validation and structure-degradation relationship analyses guided by global proteomics reveal highly selective and potent phenyl glutarimide-based degraders targeting previously uncharacterized neosubstrates such as KDM4B, G3BP2 and VCL; none of which contain the classical CRBN β-hairpin degron. These findings underscore the power of unbiased high-throughput proteomics in MGD drug discovery and reveal a substantially expanded CRBN neosubstrate landscape beyond that defined by classical immunomodulatory imid drugs (IMiDs).
    DOI:  https://doi.org/10.1038/s41467-025-62829-0
  15. Anal Chem. 2025 Aug 26.
      We developed a novel method using gas chromatography - Orbitrap mass spectrometry (GC Orbitrap MS) to measure the intramolecular carbon isotope value of pyruvic acid and acetic acid with high mass accuracy and mass resolution with nanomole (10-100) injections of analyte. Previous efforts to analyze intramolecular isotope patterns of small organic acids have been limited by labor-intensive chemical degradation steps, a narrow potential analyte pool, or large sample mass requirements. We present a new way to trap an analyte peak, which is then tested by measuring the molecular average and intramolecular carbon isotope values of pyruvic acid and acetic acid standards. Molecular average (MA) δ13C measurements with our method agreed with complementary methods within 2.2-2.6‰ relative mean standard deviation (RMSD) for 0.1 micromoles of compound, whereas intramolecular δ13C values varied from the complementary method from 1.9 to 4.9‰ RMSD. Intramolecular δ13C measurements of pyruvic acid and acetic acid yielded acquisition errors (σAE) of better than 0.2‰ and an experimental reproducibility (σER) of <2.8‰ and <3.4‰, respectively, for 0.1 micromoles of analyte. This method enables measurements of the intramolecular δ13C values of small organic acids sufficiently precise to differentiate synthesis pathways for these compounds, including for pathways within the central metabolism, and in extracts of carbonaceous meteorites, which are theorized to have formed in interstellar ices.
    DOI:  https://doi.org/10.1021/acs.analchem.5c02194
  16. J Proteome Res. 2025 Aug 27.
      Recently, several methods have been proposed for predicting peptide MS/MS fragment intensity profiles. These predicted profiles may be used for generating spectral libraries for data-independent acquisition analysis, or for improving peptide identification by rescoring the peptide-spectra matches identified by search engines such as MaxQuant. Although some of the proposed intensity prediction methods generate high quality spectral libraries and significantly improve peptide identification, they are computationally expensive and their parameters are difficult to interpret. In this paper, we introduce FastSpel (fast spectral library), a fast and interpretable fragment intensity prediction method for tryptic peptides. Testing FastSpel on 23 independent data sets, we show that its performance, in terms of improving peptide identification via rescoring and spectral library generation, is comparable with those of the existing state-of-the-art methods, while being over 2 orders of magnitude less computationally expensive than these methods. Moreover, analysis of parameters of the model corroborates known fragmentation rules, such as the "proline effect", and suggests novel patterns. In addition to FastSpel, we propose a simple scoring function that achieves rescoring/identification performance close to that of Percolator, a widely used program for this purpose, without requiring model training as Percolator does.
    Keywords:  mass spectrometry; rescoring; spectrum prediction
    DOI:  https://doi.org/10.1021/acs.jproteome.5c00279