2. Academic Validation
  3. Quantification of five intracellular and extracellular methionine pathway intermediates using stable isotope dilution UHPLC-MS/MS

Quantification of five intracellular and extracellular methionine pathway intermediates using stable isotope dilution UHPLC-MS/MS

  • J Chromatogr A. 2025 Aug 16:1755:466036. doi: 10.1016/j.chroma.2025.466036.
Lyuye Zhang 1,  Ziyu Liang 2,  Weibing Zhang 3,  Hailin Wang 4
Affiliations

Affiliations

  • 1 Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China; State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
  • 2 State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; University of Chinese Academy of Sciences, Beijing 100049, China.
  • 3 Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: weibingzhang@ecust.edu.cn.
  • 4 State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; University of Chinese Academy of Sciences, Beijing 100049, China; Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China. Electronic address: hlwang@rcees.ac.cn.
PMID: 40398135 DOI: 10.1016/j.chroma.2025.466036
Abstract

Unraveling the intricate mechanisms underlying methionine metabolism reprogramming and its extracellular release during Apoptosis requires robust and sensitive quantification of key intermediates in both intracellular and extracellular compartments. Here, we developed a highly sensitive and precise stable isotope-dilution UHPLC-MS/MS method for simultaneous quantification of five critical intermediates in methionine cycle and methionine salvage pathway: l-methionine (Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), l-homocysteine (Hcy), and 5'-methylthioadenosine (5'-MTA). Through optimizing mobile phase additives and implementing an effective dilution strategy, this method minimizes matrix effects. The recovery rates of the five compounds exceeds 71.6 % in cell cytosol and cell medium samples, under a wide concentration range. The validated method presents good precision and linearity, with limits of detection (LODs) ranging from 1.9 fmol/10⁵ cells (SAH) to 555.4 fmol/10⁵ cells (Hcy) in cytosolic fractions, and from 0.7 fmol/10⁵ cells (SAH) to 52.1 fmol/10⁵ cells (Met) in culture media. Application of this validated method to UV-induced Apoptosis in human promyelocytic leukemia HL60 cells revealed significant dynamic alterations in both intracellular and extracellular release of methionine pathway intermediates during apoptotic progression. The quantification method provides a robust tool for investigating the regulation and functions of methionine metabolism across basic research and clinical applications.

Keywords

5′-MTA; Apoptosis; Methionine metabolism; SAH; SAM; UHPLC-MS/MS.

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