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

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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.

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

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Acknowledgements

This research was supported in part by funding to J.D.R. from the U.S. National Institutes of Health (NIH) (R01CA16359-01A1) and Stand Up to Cancer (SU2C-AACR-DT0509). G.S.D. was supported by a postdoctoral fellowship (PF-15-190-01-TBE) from the American Cancer Society. W.L. was supported by NIH (CA211437).

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  1. Lewis-Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Princeton, NJ, 08544, USA

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

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Correspondence to Joshua D. Rabinowitz.

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Chen, L., Ducker, G.S., Lu, W. et al. An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate. Anal Bioanal Chem 409, 5955–5964 (2017). https://doi.org/10.1007/s00216-017-0514-4

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