Abstract
Thiols serve indispensable biochemical functions across catalysis, redox homeostasis and energy metabolism. However, profiling multiple thiols at the single-cell level remains challenging due to their trace amount and susceptibility to oxidation. Herein, we report an integrated strategy for thiol profiling at the single-cell level which combines live-cell labeling with organic mass cytometry. The live-cell labeling strategy facilitates the comprehensive measurement of intrinsic thiols with expanded coverage and improved sensitivity, while organic mass cytometry enables simultaneous quantification of 27 labeled thiols and 355 other metabolites from single cells. Assessment of metabolic fluctuation upon stimulation demonstrates practicability and accuracy of this integrated methodology which is capable of pathway activity monitoring, metabolic network mapping and untargeted metabolome profiling. Further application of this method in investigating RSL3-triggered ferroptosis reveals that RSL3 inhibits glutathione synthesis via nuclear factor E2-related factor 2- glutathione axis and results in heterogenous glutathione metabolism between subtypes.
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Data availability
The metabolomic MS raw data have been deposited to MetaboLights with the dataset identifier MTBLS13900. The data that support the findings of this study are available in the supplementary material of this article. Source data are provided with this paper.
Code availability
The code developed in this manuscript has been submitted to Code Ocean59.
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Acknowledgements
This work was financially supported by the Natural Science Foundation of China (No. 22125401 to Y.B.) and the National Key R&D Program of China (2022YFC3400700 and 2023YFF1205900 to Y.B.).
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Y.B. proposed the study concept and strategy. D.M. designed and performed the experiments and data analysis. Q.L. assisted with the data analysis of untargeted metabolome library. Y.Z. involved in the construction of organic mass cytometry. Y.W. and X.L. helped with the western blot experiments. Y.Z. and S.Q. involved in the discussion. D.M., Q.L., Y.Z., S.Q. and Y.B. wrote the manuscript.
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Nature Communications thanks Rosario M. Sanchez-Martin, who co-reviewed with Victoria Cano-Cortés, Tong Zhang, and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
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Miao, D., Li, Q., Zhang, Y. et al. Single-cell thiol profiling enabled by live-cell labeling reveals metabolic heterogeneity in ferroptosis. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70336-z
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DOI: https://doi.org/10.1038/s41467-026-70336-z
