Abstract
The analysis of metabolic perturbation in biological samples is crucial to understand mechanisms of metabolic diseases. Here, we describe a protocol for quantitative stable isotope-labeled metabolite tracing of cysteine metabolism in cultured cells. This protocol relies on an extraction protocol to derivatize free thiols to prevent oxidation. In addition, the quantitative tracing of serine into multiple pathways, including the glutathione synthesis pathway, allows for the interrogation of cysteine and glutathione synthesis. This protocol provides a flexible framework that can be adapted to interrogate many metabolites and pathways of interest.
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Acknowledgments
This work was supported by the New Faculty Startup Fund from Seoul National University and the National Research Foundation of Korea (NRF-2022M3A9I2017587, NRF-2022R1C1C1003619) to YPK. This work was supported by NIH/NCI R37CA230042 to GMD. We thank Sang Jun Yoon and Yumi Kim for critical reading of the manuscript and helpful suggestions.
Author Contributions
Y.P.K and G.M.D. wrote the manuscript.
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The authors declare no competing interests.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Kang, Y.P., DeNicola, G.M. (2023). Quantification and Tracing of Stable Isotope into Cysteine and Glutathione. In: Papa, S., Bubici, C. (eds) Metabolic Reprogramming. Methods in Molecular Biology, vol 2675. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3247-5_5
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DOI: https://doi.org/10.1007/978-1-0716-3247-5_5
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