An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate

Li Chen, Gregory S. Ducker, Wenyun Lu, Xin Teng, Joshua D. Rabinowitz

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. [Figure not available: see fulltext.].

Original languageEnglish (US)
Pages (from-to)5955-5964
Number of pages10
JournalAnalytical and Bioanalytical Chemistry
Volume409
Issue number25
DOIs
StatePublished - Oct 1 2017

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry

Keywords

  • Chemical derivatization
  • Folate
  • Interconversion
  • LC-MS
  • Methylene-THF
  • One-carbon metabolism

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