Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Correspondence
  • Published:

Discovery of metabolites prevails amid in-source fragmentation

Nature Metabolism volume 7, pages 435–437 (2025)Cite this article

Subjects

The detectable ‘dark metabolome’, or unannotated signals in untargeted metabolomics, has become a focal point of debate. The recent Correspondence by Giera et al.1 published in Nature Metabolism argued that most unannotated signals in liquid chromatography–tandem mass spectrometry (LC-MS/MS) data are in-source fragments (ISFs), and hence measurement artefacts rather than new molecules. This claim was based on the observation that 70% of ions detected from 931,000 molecular standards using collision-induced dissociation at 0 eV were ISFs, which led to the interpretation that the dark metabolome may be smaller than previously thought — or even nonexistent1,2,3. However, neither the methodological details nor the data are available to verify this claim. Media headlines, such as “The Dark Metabolome: A Figment of Our Fragmentation?”2, amplified the sensationalism. However, past research emphasize that such conclusions depend on instrumentation, tuning, sample type, matrix, extraction methods, analyte concentration and data analysis: these studies reported ISF contributions of 2–25% of all detected ions4,5. To minimize ISFs, some instruments have dedicated soft ion transfer modes, although this can reduce sensitivity.

Abundant evidence highlights the ongoing discovery of new metabolites and biochemical pathways, even in well-studied organisms like Escherichia coli, humans and mice7,8,9. Our analysis of a well-studied US National Institute of Standards and Technology (NIST) human fecal reference standard dataset revealed that, even after accounting for all ion forms, 82% of molecules lacked annotations for any ion form when grouped using retention time, MS/MS information and peak shape analysis to estimate the number of molecules they represent (Fig. 1d,e). This result underscores the enduring presence of the dark metabolome, even in thoroughly characterized organisms. The recent and steadily increasing adoption of powerful bioinformatics and data science techniques in metabolomics has propelled the discovery of new metabolites, as shown using rarefaction (Fig. 1f,g), reinforcing the existence of a sizable dark metabolome that awaits discovery.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: ISF distributions, effect on the dark metabolome, and yearly growth of new annotations.

Data availability

The datasets re-analysed here were downloaded from Metabolomics Workbench (study ST002336), MassIVE/Global Natural Product Social Molecular Networking 2 (GNPS accession codes MSV000080673 and MSV000093526) and Zenodo (accession code 13890851).

Code availability

Annotated code used for data analyses performed in this Correspondence, with instructions for use, can be found on GitHub11,12.

References

  1. Giera, M., Aisporna, A., Uritboonthai, W. & Siuzdak, G. Nat. Metab. 6, 1647–1648 (2024).

  2. Strachan, J. The dark metabolome: a figment of our fragmentation? Analytical Scientist https://theanalyticalscientist.com/fields-applications/the-dark-metabolome-a-figment-of-our-fragmentation (2024).

  3. Lowe, D. Phantom metabolites? Science https://www.science.org/content/blog-post/phantom-metabolites (2024).

  4. Schmid, R. et al. Nat. Commun. 12, 3832 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lu, W. et al. Anal. Chem. 92, 11573–11581 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Xu, Y.-F., Lu, W. & Rabinowitz, J. D. Anal. Chem. 87, 2273–2281 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Agongo, J. et al. Anal. Chem. 96, 11639–11643 (2024).

    Article  CAS  PubMed  Google Scholar 

  8. Qiang, H. et al. Preprint at bioRxiv https://doi.org/10.1101/2024.11.13.623458 (2024).

  9. Gentry, E. C. et al. Nature 626, 419–426 (2024).

    Article  CAS  PubMed  Google Scholar 

  10. Baygi, S. F., Kumar, Y. & Barupal, D. K. Anal. Chem. 95, 9480–9487 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rutz, A. et al. GitHub https://github.com/zamboni-lab/ion-type-analysis/tree/correspondence (2024).

  12. El Abiead, Y. et al. GitHub https://github.com/YasinEl/DarkMetabolome_figures/blob/correspondence (2024).

Download references

Acknowledgements

We acknowledge the NIST Complex Microbial Systems Group for providing the NIST material. A.M.C.-R. and P.C.D. were supported by the Gordon and Betty Moore Foundation, GBMF12120.

Author information

Author notes

  1. These authors contributed equally: Yasin El Abiead, Adriano Rutz.

Authors and Affiliations

  1. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA

    Yasin El Abiead, Simone Zuffa, Shipei Xing, Andres Mauricio Caraballo-Rodriguez, Helena Mannochio-Russo, Ipsita Mohanty & Pieter C. Dorrestein

  2. Institute for Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

    Adriano Rutz, Mario S. P. Correia & Nicola Zamboni

  3. Thermo Fisher Scientific, San Jose, CA, USA

    Bashar Amer

  4. Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic

    Corinna Brungs, Robin Schmid & Tomáš Pluskal

  5. Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA

    Andres Mauricio Caraballo-Rodriguez & Pieter C. Dorrestein

  6. Division of Gastroenterology, University of California, San Diego, La Jolla, CA, USA

    Amir Zarrinpar

  7. Division of Gastroenterology, Jennifer Moreno Department of Veterans Affairs Medical Center, La Jolla, CA, USA

    Amir Zarrinpar

  8. Shu Chien-Gene Lay Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

    Amir Zarrinpar & Rob Knight

  9. Institute of Diabetes and Metabolic Health, University of California, San Diego, La Jolla, CA, USA

    Amir Zarrinpar

  10. Metabolomics and Proteomics Core, Helmholtz Zentrum München, Neuherberg, Germany

    Michael Witting

  11. Chair of Analytical Food Chemistry, TUM School of Life Sciences, Technical University of Munich, Freising-Weihenstephan, Germany

    Michael Witting

  12. Department of Computer Science, University of Antwerp, Antwerp, Belgium

    Wout Bittremieux

  13. Center for Microbiome Innovation, University of California San Diego, La Jolla, CA, USA

    Rob Knight & Pieter C. Dorrestein

  14. Department of Biochemistry and Molecular Biology and Department of Veterinary and Biomedical Sciences, the Pennsylvania State University, University Park, PA, USA

    Andrew D. Patterson

  15. Bioinformatics Group, Wageningen University, Wageningen, the Netherlands

    Justin J. J. van der Hooft

  16. Department of Biochemistry, University of Johannesburg, Johannesburg, South Africa

    Justin J. J. van der Hooft

  17. Institute of Computer Science, Friedrich Schiller University Jena, Jena, Germany

    Sebastian Böcker

  18. Department of Biochemistry, Cell and Systems Biology, Centre for Metabolomics Research, Institute of Systems, Molecular, and Integrative Biology, University of Liverpool, Liverpool, UK

    Warwick B. Dunn

  19. Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada

    Roger G. Linington

  20. Department of Biological Science, University of Alberta, Edmonton, Alberta, Canada

    David S. Wishart

  21. Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland

    Jean-Luc Wolfender

  22. School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland

    Jean-Luc Wolfender

  23. West Coast Metabolomics Center, University of California, Davis, Davis, CA, USA

    Oliver Fiehn

  24. Department of Pharmacology, University of California San Diego, La Jolla, CA, USA

    Pieter C. Dorrestein

Authors
  1. Yasin El Abiead
    View author publications

    Search author on:PubMed Google Scholar

  2. Adriano Rutz
    View author publications

    Search author on:PubMed Google Scholar

  3. Simone Zuffa
    View author publications

    Search author on:PubMed Google Scholar

  4. Bashar Amer
    View author publications

    Search author on:PubMed Google Scholar

  5. Shipei Xing
    View author publications

    Search author on:PubMed Google Scholar

  6. Corinna Brungs
    View author publications

    Search author on:PubMed Google Scholar

  7. Robin Schmid
    View author publications

    Search author on:PubMed Google Scholar

  8. Mario S. P. Correia
    View author publications

    Search author on:PubMed Google Scholar

  9. Andres Mauricio Caraballo-Rodriguez
    View author publications

    Search author on:PubMed Google Scholar

  10. Amir Zarrinpar
    View author publications

    Search author on:PubMed Google Scholar

  11. Helena Mannochio-Russo
    View author publications

    Search author on:PubMed Google Scholar

  12. Michael Witting
    View author publications

    Search author on:PubMed Google Scholar

  13. Ipsita Mohanty
    View author publications

    Search author on:PubMed Google Scholar

  14. Tomáš Pluskal
    View author publications

    Search author on:PubMed Google Scholar

  15. Wout Bittremieux
    View author publications

    Search author on:PubMed Google Scholar

  16. Rob Knight
    View author publications

    Search author on:PubMed Google Scholar

  17. Andrew D. Patterson
    View author publications

    Search author on:PubMed Google Scholar

  18. Justin J. J. van der Hooft
    View author publications

    Search author on:PubMed Google Scholar

  19. Sebastian Böcker
    View author publications

    Search author on:PubMed Google Scholar

  20. Warwick B. Dunn
    View author publications

    Search author on:PubMed Google Scholar

  21. Roger G. Linington
    View author publications

    Search author on:PubMed Google Scholar

  22. David S. Wishart
    View author publications

    Search author on:PubMed Google Scholar

  23. Jean-Luc Wolfender
    View author publications

    Search author on:PubMed Google Scholar

  24. Oliver Fiehn
    View author publications

    Search author on:PubMed Google Scholar

  25. Nicola Zamboni
    View author publications

    Search author on:PubMed Google Scholar

  26. Pieter C. Dorrestein
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Pieter C. Dorrestein.

Ethics declarations

Competing interests

P.C.D. is an advisor to and holds equity in Cybele, BileOmix and Sirenas and is a scientific co-founder of, is advisor to, holds equity in and/or received income from Ometa, Enveda and Arome with prior approval by the University of California, San Diego. P.C.D. also consulted for DSM Animal Health in 2023. J.J.J.v.d.H. is member of the scientific advisory board of Naicons Srl., Milano, Italy and consults for Corteva Agriscience, Indianapolis, IN, USA. R.S. and T.P. are co-founders of mzio GmbH. A.Z. is a co-founder of and equity holder in Endure Biotherapeutics. R.K. is a scientific advisory board member and consultant for BiomeSense, Inc., has equity and receives income. He is a scientific advisory board member and has equity in GenCirq. He is a consultant for DayTwo and receives income. He has equity in and acts as a consultant for Cybele. He is a co-founder of Biota, Inc., and has equity. He is a cofounder of Micronoma, has equity and is a scientific advisory board member. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. The other authors declare no competing interests. S.B. is co-founder of Bright Giant.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

El Abiead, Y., Rutz, A., Zuffa, S. et al. Discovery of metabolites prevails amid in-source fragmentation. Nat Metab 7, 435–437 (2025). https://doi.org/10.1038/s42255-025-01239-4

Download citation

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s42255-025-01239-4

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research