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

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

Keywords: Chemical derivatization; Folate; Interconversion; LC-MS; Methylene-THF; One-carbon metabolism.

Figure 1. Tetrahydrofolate (THF) structure and interconversion

(a) Structure of tetrahydrofolate. (b) Scheme of intracellular reactions, spontaneous reactions and chemical derivatization of THF species.

Figure 2. LC-MS measurement of THF species from cultured cells

(a) Schematic of analytical workflow. (b) Extracted ion chromatograms (indicated exact mass ± 10 ppm) of THF, methylene-THF, methenyl-THF, 5/10-formyl-THF and 5-methyl-THF.

Figure 3. Residual enzymatic conversion of 5-formyl-THF to methenyl-THF in lysates

(a) Reaction schematic for methenyl-THF, 10-formyl- and 5-formyl-THF interconversion. The proton highlighted in red can exchange with H2O. (b) Labeling kinetics of freshly dissolved unlabeled methenyl-THF standard incubated in D2O phosphate buffer (pH 7) at 4°C. To verify that 5-formyl-THF, unlike 10-formyl-THF, does not interconvert with methenyl-THF under these conditions, an identical experiment was performed with 5-formyl-THF standard (Mean ± SD, N = 3). (c) Folate and metabolite labeling patterns after 24 h incubation of in HEK293T cells with 1 mM [13C,2H]formate in the growth media (Mean ± SD, N ≥ 2). (d) Conversion of 5-formyl-THF to methenyl-THF in cell extracts. HEK293T cell extracts were prepared as described in method, and optionally heated to 60°C for 5 min after addition (when indicated) of 4 pmol 5-formyl-THF standard (Mean ± SD, N = 3).

Figure 4. Methylene-THF’s 1C unit exchanges with formaldehyde

(a) Reaction schematic for THF and methylene-THF interconversion. The methylene group highlighted in red can exchange with formaldehyde. (b) Labeling patterns in HEK293T cells after 24 h incubation with [3-13C]serine. Note the lack of labeling of methylene-THF, despite labeling of its downstream products, indicative of label loss during the sample preparation process (Mean ± SD, N = 3). (c) Labeling patterns in HEK293T cells after 24 h incubation with 1 mM [13C,2H]formate (Mean ± SD, N = 3). (d) Labeling kinetics of freshly dissolved unlabeled THF or methylene-THF standard incubated in 1:1 H2O:[U-2H]MeOH at 4°C. (e) Labeling pattern of freshly dissolved unlabeled methylene-THF standard incubated at 4°C for 2 h in unlabeled H2O:MeOH with the indicated concentration of deuterated formaldehyde.

Figure 5. Quantification of methylene-THF via NaBD3CN reduction

(a) Reduction of methylene-THF to 5-methyl-THF with NaBH3CN (or NaBD3CN). The hydrogen highlighted in red comes from the reducing reagent. (b) Change in 5-methyl-THF signal upon NaBH3CN reduction of cell extracts. (c) Change in 5-methyl-THF signal upon NaBD3CN reduction of cell extracts. Data in (b) and (c) are mean ± SD, N = 3. (d) 5-methyl-THF labeling after NaBD3CN reduction of the indicated folate standards. DHF + HCHO indicates DHF standard incubated with 0.1% unlabeled formaldehyde (Mean ± SD, N ≥ 2).

Figure 6

Acute effect of methotrexate on cellular folates. HEK293T cells were incubated with 1 μM methotrexate for 2 h before extraction. Pool sizes are normalized to DMSO-treated control cells (Mean ± SD, N ≥ 3).