Charting the Undiscovered Metabolome with Synthetic Multiplexing

Patan, A.; Xing, S.; Charron-Lamoureux, V.; Hu, Z.; Deleray, V.; Agongo, J.; El Abiead, Y.; Mannochio-Russo, H.; Mohanty, I.; Gouda, H.; Zemlin, J.; Rajkumar, P.; Lee, C.; Leanos, D.; Weimann, N.; Tsuda, W.; Giddings, S.; Bui, T.; Kvitne, K. E.; Zhao, H. N.; Zuffa, S.; Nguyen, V.; Andrade, A.; Goncalves Nunes, W. D.; Caraballo-Rodriguez, A. M.; Caetano David, L.; Carver, J.; Bandeira, N.; Wang, M.; Burnett, L.; Siegel, D.; Dorrestein, P. C.

doi:10.1101/2025.11.18.689170

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bioRxiv · 10.1101/2025.11.18.689170

Charting the Undiscovered Metabolome with Synthetic Multiplexing

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Abstract

Most molecular features detected in untargeted metabolomics remain uncharacterized due to the limited scope of existing spectral reference libraries. We synthesized >100,000 biologically inspired compounds using multiplexed reactions, of which 91% were absent from existing structural databases, and searched the resulting MS/MS library across >1.7 billion public spectra, increasing annotation rates by 17.4%. This approach revealed previously undescribed exposure-derived metabolites, including ibuprofen-carnitine. Because ibuprofen has been linked to rhabdomyolysis, reduced mitochondrial function, and impaired muscle recovery in carnitine-limited contexts, we investigated the functional relevance of this conjugate. Ibuprofen-carnitine reduced carnitine transport via the OCTN2 transporter, and in a postpartum mouse muscle injury model, ibuprofen delayed muscle repair that could be rescued by carnitine supplementation, with urinary ibuprofen-carnitine:carnitine ratios tracking this effect. These findings support a hypothesis whereby NSAID-carnitine conjugates compete for carnitine transport, impairing energy metabolism and muscle recovery in susceptible individuals. Synthetic multiplexing thus provides a scalable route to annotate the dark metabolome and generate experimentally testable biological hypotheses.

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Patan, A., Xing, S., Charron-Lamoureux, V., Hu, Z., Deleray, V., Agongo, J., El Abiead, Y., Mannochio-Russo, H., Mohanty, I., Gouda, H., Zemlin, J., Rajkumar, P., Lee, C., Leanos, D., Weimann, N., Tsuda, W., Giddings, S., Bui, T., Kvitne, K. E., Zhao, H. N., Zuffa, S., Nguyen, V., Andrade, A., Goncalves Nunes, W. D., Caraballo-Rodriguez, A. M., Caetano David, L., Carver, J., Bandeira, N., Wang, M., Burnett, L., Siegel, D., Dorrestein, P. C.. 2025-11-19. Charting the Undiscovered Metabolome with Synthetic Multiplexing. https://doi.org/10.1101/2025.11.18.689170

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