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

作者：Chen Li; Ducker Gregory S; Lu Wenyun; Teng Xin; Rabinowitz Joshua D^*

来源：Analytical and Bioanalytical Chemistry, 2017, 409(25): 5955-5964.

DOI：10.1007/s00216-017-0514-4

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

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.

• 出版日期2017-10