bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2024–07–21
fourteen papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh
  1. Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics.
  2. Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase.
  3. PeakQC: A Software Tool for Omics-Agnostic Automated Quality Control of Mass Spectrometry Data.
  4. A hybrid DDA/DIA-PASEF based assay library for a deep proteotyping of triple-negative breast cancer.
  5. Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.
  6. Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches.
  7. Ketogenesis supports hepatic polyunsaturated fatty acid homeostasis via fatty acid elongation.
  8. Analysis and Quantification of the Mitochondrial-ER Lipidome.
  9. Full Native timsTOF PASEF-Enabled Quantitative Proteomics with the i2MassChroQ Software Package.
  10. ICP-MS Analysis of Iron from Biological Samples.
  11. Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.
  12. Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility-Mass Spectrometry.
  13. MetaboLink: A web application for Streamlined Processing and Analysis of Large-Scale Untargeted Metabolomics Data.
  14. FragHub: A Mass Spectral Library Data Integration Workflow.
  1. Anal Chim Acta. 2024 Aug 15. pii: S0003-2670(24)00714-1. [Epub ahead of print]1317 342913
    Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics.
    Johannes Scholz, Edward Rudt, Anna Gremme, Carina M Gaßmöller Née Wienken, Julia Bornhorst, Heiko Hayen.
       BACKGROUND: Lipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations.
    RESULTS: A SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome.
    SIGNIFICANCE: This method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.
    Keywords:  Caenorhabditis elegans; Ceramides; Lipidomics; SFC-TIMS-MS; Sphingolipids
    DOI:  https://doi.org/10.1016/j.aca.2024.342913
  2. Metabolomics. 2024 Jul 16. 20(4): 77
    Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase.
    Francesca Day, Justin O'Sullivan, Farha Ramzan, Chris Pook.
       INTRODUCTION: Accurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography-mass spectrometry are predominantly used to target polar metabolites.
    OBJECTIVES: This study aims to demonstrate a simple one-step extraction combined with liquid chromatography-mass spectrometry (LC-MS) that reliably retains polar metabolites.
    METHODS: The method involves a MilliQ + 10% trichloroacetic acid extraction from 6 healthy individuals serum, combined with porous graphitic carbon liquid chromatography-mass spectrometry (LC-MS). The coefficient of variation (CV) assessed retention reliability of polar metabolites with logP as low as - 9. QreSS (Quantification, Retention, and System Suitability) internal standards determined the method's consistency and recovery efficiency.
    RESULTS: The method demonstrated reliable retention (CV < 0.30) of polar metabolites within a logP range of - 9.1 to 5.6. QreSS internal standards confirmed consistent performance (CV < 0.16) and effective recovery (70-130%) of polar to mid-polar metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson's correlation coefficient > 0.80) within their concentration range. Repeatability was demonstrated through clustering of repeated extractions from a single sample.
    CONCLUSION: This LC-MS method is better suited to covering the polar segment of the metabolome than current methods, offering a reliable and efficient approach for accurate quantification of polar metabolites in untargeted metabolomics.
    Keywords:  HILIC; LC–MS; Polar metabolites; Porous graphitic carbon; Trichloroacetic acid; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/s11306-024-02146-7
  3. J Am Soc Mass Spectrom. 2024 Jul 16.
    PeakQC: A Software Tool for Omics-Agnostic Automated Quality Control of Mass Spectrometry Data.
    Andrea Harrison, Josie G Eder, Priscila M Lalli, Nathalie Munoz, Yuqian Gao, Chaevien S Clendinen, Daniel J Orton, Xueyun Zheng, Sarah M Williams, Sneha P Couvillion, Rosalie K Chu, Vimal K Balasubramanian, Arunima Bhattacharjee, Christopher R Anderton, Kyle R Pomraning, Kristin E Burnum-Johnson, Tao Liu, Jennifer E Kyle, Aivett Bilbao.
      Mass spectrometry is broadly employed to study complex molecular mechanisms in various biological and environmental fields, enabling 'omics' research such as proteomics, metabolomics, and lipidomics. As study cohorts grow larger and more complex with dozens to hundreds of samples, the need for robust quality control (QC) measures through automated software tools becomes paramount to ensure the integrity, high quality, and validity of scientific conclusions from downstream analyses and minimize the waste of resources. Since existing QC tools are mostly dedicated to proteomics, automated solutions supporting metabolomics are needed. To address this need, we developed the software PeakQC, a tool for automated QC of MS data that is independent of omics molecular types (i.e., omics-agnostic). It allows automated extraction and inspection of peak metrics of precursor ions (e.g., errors in mass, retention time, arrival time) and supports various instrumentations and acquisition types, from infusion experiments or using liquid chromatography and/or ion mobility spectrometry front-end separations and with/without fragmentation spectra from data-dependent or independent acquisition analyses. Diagnostic plots for fragmentation spectra are also generated. Here, we describe and illustrate PeakQC's functionalities using different representative data sets, demonstrating its utility as a valuable tool for enhancing the quality and reliability of omics mass spectrometry analyses.
    Keywords:  computational tool; data processing; data-dependent acquisition; data-independent acquisition, data quality control, proteomics; ion mobility spectrometry; lipidomics, algorithm; liquid chromatography; metabolomics
    DOI:  https://doi.org/10.1021/jasms.4c00146
  4. Sci Data. 2024 Jul 18. 11(1): 794
    A hybrid DDA/DIA-PASEF based assay library for a deep proteotyping of triple-negative breast cancer.
    Petr Lapcik, Klara Synkova, Lucia Janacova, Pavla Bouchalova, David Potesil, Rudolf Nenutil, Pavel Bouchal.
      Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and deeper proteome coverage is needed for its molecular characterization. We present comprehensive library of targeted mass spectrometry assays specific for TNBC and demonstrate its applicability. Proteins were extracted from 105 TNBC tissues and digested. Aliquots were pooled, fractionated using hydrophilic chromatography and analyzed by LC-MS/MS in data-dependent acquisition (DDA) parallel accumulation-serial fragmentation (PASEF) mode on timsTOF Pro LC-MS system. 16 individual lysates were analyzed in data-independent acquisition (DIA)-PASEF mode. Hybrid library was generated in Spectronaut software and covers 244,464 precursors, 168,006 peptides and 11,564 protein groups (FDR = 1%). Application of our library for pilot quantitative analysis of 16 tissues increased identification numbers in Spectronaut 18.5 and DIA-NN 1.8.1 software compared to library-free setting, with Spectronaut achieving the best results represented by 190,310 precursors, 140,566 peptides, and 10,463 protein groups. In conclusion, we introduce assay library that offers the deepest coverage of TNBC proteome to date. The TNBC library is available via PRIDE repository (PXD047793).
    DOI:  https://doi.org/10.1038/s41597-024-03632-2
  5. Nat Commun. 2024 Jul 13. 15(1): 5890
    Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.
    Meera Gupta, Alex N T Johnson, Edward R Cruz, Eli J Costa, Randi L Guest, Sophia Hsin-Jung Li, Elizabeth M Hart, Thao Nguyen, Michael Stadlmeier, Benjamin P Bratton, Thomas J Silhavy, Ned S Wingreen, Zemer Gitai, Martin Wühr.
      Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.
    DOI:  https://doi.org/10.1038/s41467-024-49920-8
  6. Methods Mol Biol. 2024 ;2839 113-130
    Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches.
    Ronivaldo Rodrigues da Silva, Victoria Adedoyin, Jennifer L DuBois.
      Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generally nonpathogenic anaerobe and heme auxotroph.
    Keywords:  Anaerobe; Bacteria; Bacteroides; Heme metabolism; LC-MS; Metabolomics; Microbiome; Stable isotope
    DOI:  https://doi.org/10.1007/978-1-0716-4043-2_7
  7. bioRxiv. 2024 Jul 11. pii: 2024.07.09.602593. [Epub ahead of print]
    Ketogenesis supports hepatic polyunsaturated fatty acid homeostasis via fatty acid elongation.
    Eric D Queathem, Zahra Moazzami, David B Stagg, Alisa B Nelson, Kyle Fulghum, Abdirahman Hayir, Alisha Seay, Jacob R Gillingham, D Andre d'Avignon, Xianlin Han, Hai-Bin Ruan, Peter A Crawford, Patrycja Puchalska.
      Therapeutic interventions targeting hepatic lipid metabolism in metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) remain elusive. Using mass spectrometry-based stable isotope tracing and shotgun lipidomics, we established a novel link between ketogenesis and MASLD pathophysiology. Our findings show that mouse liver and primary hepatocytes consume ketone bodies to support fatty acid (FA) biosynthesis via both de novo lipogenesis (DNL) and FA elongation. Analysis of 13 C-labeled FAs in hepatocytes lacking mitochondrial D-β-hydroxybutyrate dehydrogenase (BDH1) revealed a partial reliance on mitochondrial conversion of D-βOHB to acetoacetate (AcAc) for cytoplasmic DNL contribution, whereas FA elongation from ketone bodies was fully dependent on cytosolic acetoacetyl-CoA synthetase (AACS). Ketone bodies were essential for polyunsaturated FA (PUFA) homeostasis in hepatocytes, as loss of AACS diminished both free and esterified PUFAs. Ketogenic insufficiency depleted liver PUFAs and increased triacylglycerols, mimicking human MASLD, suggesting that ketogenesis supports PUFA homeostasis, and may mitigate MASLD-MASH progression in humans.
    DOI:  https://doi.org/10.1101/2024.07.09.602593
  8. Bio Protoc. 2024 Jul 05. 14(13): e5028
    Analysis and Quantification of the Mitochondrial-ER Lipidome.
    Alexis R Diaz-Vegas, Anthony S Don, James G Burchfield.
      Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress. Key features • Analysis and quantification of lipids in mitochondria-ER fraction through liquid chromatography-tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics). • LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems. • LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.
    Keywords:  Cardiolipin; Ceramides; Endoplasmic reticulum; Lipidomics; Mitochondria; Subcellular fractionation
    DOI:  https://doi.org/10.21769/BioProtoc.5028
  9. J Proteome Res. 2024 Jul 17.
    Full Native timsTOF PASEF-Enabled Quantitative Proteomics with the i2MassChroQ Software Package.
    Olivier Langella, Thomas Renne, Thierry Balliau, Marlène Davanture, Sven Brehmer, Michel Zivy, Mélisande Blein-Nicolas, Filippo Rusconi.
      Ion mobility mass spectrometry has become popular in proteomics lately, in particular because the Bruker timsTOF instruments have found significant adoption in proteomics facilities. The Bruker's implementation of the ion mobility dimension generates massive amounts of mass spectrometric data that require carefully designed software both to extract meaningful information and to perform processing tasks at reasonable speed. In a historical move, the Bruker company decided to harness the skills of the scientific software development community by releasing to the public the timsTOF data file format specification. As a proteomics facility that has been developing Free Open Source Software (FOSS) solutions since decades, we took advantage of this opportunity to implement the very first FOSS proteomics complete solution to natively read the timsTOF data, low-level process them, and explore them in an integrated quantitative proteomics software environment. We dubbed our software i2MassChroQ because it implements a (peptide)identification-(protein)inference-mass-chromatogram-quantification processing workflow. The software benchmarking results reported in this paper show that i2MassChroQ performed better than competing software on two critical characteristics: (1) feature extraction capability and (2) protein quantitative dynamic range. Altogether, i2MassChroQ yielded better quantified protein numbers, both in a technical replicate MS runs setting and in a differential protein abundance analysis setting.
    Keywords:  C++; DDA bottom-up proteomics; Free Software; ion mobility; mass spectrometry; quantitative proteomics; software; timsTOF
    DOI:  https://doi.org/10.1021/acs.jproteome.3c00732
  10. Methods Mol Biol. 2024 ;2839 31-41
    ICP-MS Analysis of Iron from Biological Samples.
    Javier Seravalli.
      Elemental analysis can provide trace concentrations of iron and other transition elements at nanomolar (μg/L) concentrations in whole bacterial and mammalian cells, subcellular compartments, biological fluids, and tissues. The best method of analysis is by far Inductively Coupled Plasma Mass Spectrometry (ICP-MS). I describe here a very general method for the sample preparation, instrument settings, method development, and analysis. The method can be extended to up to 20 common elements in biological samples.
    Keywords:  Elemental analysis; Hydrogen peroxide; ICP-MS; Inductively coupled plasma; Iron; Mass spectrometry; Nitric acid
    DOI:  https://doi.org/10.1007/978-1-0716-4043-2_2
  11. J Extracell Vesicles. 2024 Jul;13(7): e12449
    Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.
    Joël E J Beaumont, Lydie M O Barbeau, Jinzhe Ju, Kim G Savelkouls, Freek G Bouwman, Marijke I Zonneveld, Annelies Bronckaers, Kim R Kampen, Tom G H Keulers, Kasper M A Rouschop.
      Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.
    Keywords:  angiogenesis; extracellular vesicles; hypoxia; metabolism
    DOI:  https://doi.org/10.1002/jev2.12449
  12. J Am Chem Soc. 2024 Jul 16.
    Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility-Mass Spectrometry.
    Quentin Duez, Jeroen van de Wiel, Bob van Sluijs, Souvik Ghosh, Mathieu G Baltussen, Max T G M Derks, Jana Roithová, Wilhelm T S Huck.
      The forward design of in vitro enzymatic reaction networks (ERNs) requires a detailed analysis of network kinetics and potentially hidden interactions between the substrates and enzymes. Although flow chemistry allows for a systematic exploration of how the networks adapt to continuously changing conditions, the analysis of the reaction products is often a bottleneck. Here, we report on the interface between a continuous stirred-tank reactor, in which an immobilized enzymatic network made of 12 enzymes is compartmentalized, and an ion mobility-mass spectrometer. Feeding uniformly 13C-labeled inputs to the enzymatic network generates all isotopically labeled reaction intermediates and products, which are individually detected by ion mobility-mass spectrometry (IMS-MS) based on their mass-to-charge ratios and inverse ion mobilities. The metabolic flux can be continuously and quantitatively monitored by diluting the ERN output with nonlabeled standards of known concentrations. The real-time quantitative data obtained by IMS-MS are then harnessed to train a model of network kinetics, which proves sufficiently predictive to control the ERN output after a single optimally designed experiment. The high resolution of the time-course data provided by this approach is an important stepping stone to design and control sizable and intricate ERNs.
    DOI:  https://doi.org/10.1021/jacs.4c04218
  13. Bioinformatics. 2024 Jul 17. pii: btae459. [Epub ahead of print]
    MetaboLink: A web application for Streamlined Processing and Analysis of Large-Scale Untargeted Metabolomics Data.
    Ana Mendes, Jesper Foged Havelund, Jonas Lemvig, Veit Schwämmle, Nils J Færgeman.
       MOTIVATION: The post-processing and analysis of large-scale untargeted metabolomics data face significant challenges due to the intricate nature of correction, filtration, imputation, and normalization steps. Manual execution across various applications often leads to inefficiencies, human-induced errors, and inconsistencies within the workflow.
    RESULTS: Addressing these issues, we introduce MetaboLink, a novel web application designed to process LC-MS metabolomics datasets combining established methodologies and offering flexibility and ease of implementation. It offers visualization options for data interpretation, an interface for statistical testing, and integration with PolySTest for further tests and with VSClust for clustering analysis.
    AVAILABILITY: Fully functional tool is publicly available at https://computproteomics.bmb.sdu.dk/Metabolomics/. The source code is available at https://github.com/anitamnd/MetaboLink and a detailed description of the app can be found at https://github.com/anitamnd/MetaboLink/wiki. A tutorial video can be found at https://youtu.be/ZM6j10S6Z8Q.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btae459
  14. Anal Chem. 2024 Jul 19.
    FragHub: A Mass Spectral Library Data Integration Workflow.
    Axel Dablanc, Solweig Hennechart, Amélie Perez, Guillaume Cabanac, Yann Guitton, Nils Paulhe, Bernard Lyan, Emilien L Jamin, Franck Giacomoni, Guillaume Marti.
      Open mass spectral libraries (OMSLs) are critical for metabolite annotation and machine learning, especially given the rising volume of untargeted metabolomic studies and the development of annotation pipelines. Despite their importance, the practical application of OMSLs is hampered by the lack of standardized file formats, metadata fields, and supporting ontology. Current libraries, often restricted to specific topics or matrices, such as natural products, lipids, or the human metabolome, may limit the discovery potential of untargeted studies. The goal of FragHub is to provide users with the capability to integrate various OMSLs into a single unified format, thereby enhancing the annotation accuracy and reliability. FragHub addresses these challenges by integrating multiple OMSLs into a single comprehensive database, supporting various data formats, and harmonizing metadata. It also proposes some generic filters for the mass spectrum using a graphical user interface. Additionally, a workflow to generate in-house libraries compatible with FragHub is proposed. FragHub dynamically segregates libraries based on ionization modes and chromatography techniques, thereby enhancing data utility in metabolomic research. The FragHub Python code is publicly available under a MIT license, at the following repository: https://github.com/eMetaboHUB/FragHub. Generated data can be accessed at 10.5281/zenodo.11057687.
    DOI:  https://doi.org/10.1021/acs.analchem.4c02219
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