TitleAn LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate.
Publication TypeJournal Article
Year of Publication2017
AuthorsChen, L, Ducker, GS, Lu, W, Teng, X, Rabinowitz, JD
JournalAnal Bioanal Chem
Date Published2017 Aug 10

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. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

Alternate JournalAnal Bioanal Chem
PubMed ID28799108
Grant ListR01 CA163591 / CA / NCI NIH HHS / United States
R50 CA211437 / CA / NCI NIH HHS / United States