We mined 1.32 billion MS/MS spectra across public metabolomics repositories using reverse spectral searching and delta-mass infererence to map metabolite conjugations. We generated structural hypotheses for 24,227,439 spectra, encompassing 217,291 substructure pairs with dual spectral support and 3,412,720 additional candidates with single-match support. These results provide a pan-repository map of potential conjugation chemistry, establish a resource for structural discovery, and offer a framework to further explore the scope and diversity of the conjugated metabolome.
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Reverse spectral search is an MS/MS similarity framework originally proposed for spectral identification and later extended to improve robustness to chimeric spectra. Here, we repurpose reverse spectral searching for substructure annotation.
Two implementations of reverse cosine similarity were provided:
- matchms-based reverse cosine: revcos.py
- Flash reverse cosine: flash_cos.py
In both implementations, both the similarity score and the number of matched peaks will be output. Codes were tested using Python 3.10 on macOS (14.6, M2 Max) and Linux (Ubuntu 20.04). Packages of numpy and numba are required. For the matchms-based implementation, each spectral comparison takes several milliseconds.
- Conjugate search results of 149.9 million clustered MS/MS: Zenodo link
- GNPS conjugated metabolome libraries: GNPS external library link
- Web app to explore conjugated metabolome results: Web app link
- Datasets
- MSV000086131 (Mammalian feces)
- MSV000082261 (Diabetes study)
- MSV000082433 (Human feces, losartan conjugates)
- MSV000083306 (Tomato seedling extracts)
- MSV000098638 (Microbial cultures)
- MSV000099690 (Chemical synthesis & RT matching)
- MSV000096359 (Human urine cohort for drug conjugates)
- MSV000097935 (Human serum samples for drug conjugates)
- MSV000099690 (Retention time matching)
- MS/MS reference libraries
- GNPS (ALL_GNPS_NO_PROPOGATED.msp, downloaded on Nov 11, 2024)
- MassBank (MassBank_NIST.msp, 2024.06 release)
- MoNA (MoNA-export-LC-MS-MS_Spectra.msp, downloaded on Oct 17, 2024)
- NIST20 (Commercially available)
Xing, S. et al. Navigating the conjugated metabolome. Preprint at https://doi.org/10.64898/2026.02.06.704496 (2026).
This work is licensed under the Apache License 2.0.