Quantification and Tracing of Stable Isotope into Cysteine and Glutathione

Yun Pyo Kang; Gina M DeNicola

doi:10.1007/978-1-0716-3247-5_5

Quantification and Tracing of Stable Isotope into Cysteine and Glutathione

Methods Mol Biol. 2023:2675:51-63. doi: 10.1007/978-1-0716-3247-5_5.

Authors

Yun Pyo Kang¹, Gina M DeNicola²

Affiliations

¹ College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea.
² Department of Metabolism & Physiology, H. Lee. Moffitt Cancer Center, Tampa, FL, USA. Gina.DeNicola@Moffitt.org.

PMID: 37258755
DOI: 10.1007/978-1-0716-3247-5_5

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.

Keywords: Cell culture; Cysteine; Mass spectrometry; Quantification; Serine; Stable isotope standard; Stable isotope tracing.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Carbon Isotopes
Cells, Cultured
Cysteine*
Isotope Labeling / methods
Sulfhydryl Compounds* / metabolism

Substances

Cysteine
Sulfhydryl Compounds
Carbon Isotopes

Grants and funding

R37 CA230042/CA/NCI NIH HHS/United States