DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

Li Liu; Yu-Han Lu; Min-Dan Wang; Qun-Fei Zhao; Xiu-Ping Chen; Hang Yin; Chen-Guo Feng; Fang Zhang

doi:10.1016/j.jpha.2023.08.016

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

J Pharm Anal. 2023 Nov;13(11):1365-1373. doi: 10.1016/j.jpha.2023.08.016. Epub 2023 Sep 22.

Authors

Li Liu^{1

2}, Yu-Han Lu^{1

3}, Min-Dan Wang¹, Qun-Fei Zhao¹, Xiu-Ping Chen¹, Hang Yin², Chen-Guo Feng¹, Fang Zhang¹

Affiliations

¹ The Research Center of Chiral Drugs, Shanghai Frontiers Science Center for TCM Chemical Biology, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
² Department of Thoracic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200127, China.
³ School of Public Health, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.

Abstract

In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R² = 0.9981-0.9999), precision (interday precision of 1.6%-19.0% and intraday precision of 1.4%-19.8%) and accuracy (83.4%-115.7%). Moreover, the recovery assessments in tissues (82.5%-107.3%) and in cells (98.1%-118.9%) with GSH-¹³C2, ¹⁵N, and Cys-¹⁵N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.

Keywords: DMMIC derivatization; Esophageal squamous cell carcinoma; Glutathione anabolic pathway; KYSE-150cell; LC-MS; Metabolite profiling; p-Hydroxycinnamaldehyde.