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. 2018 Jun 19;90(12):7349-7356.
doi: 10.1021/acs.analchem.8b00650. Epub 2018 Jun 5.

Quantification of Cellular Folate Species by LC-MS After Stabilization by Derivatization

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Free PMC article

Quantification of Cellular Folate Species by LC-MS After Stabilization by Derivatization

Matthias Schittmayer et al. Anal Chem. .
Free PMC article

Abstract

Folate cofactors play a key role in one-carbon metabolism. Analysis of individual folate species is hampered by the low chemical stability and high interconvertibility of folates, which can lead to severe experimental bias. Here, we present a complete workflow that employs simultaneous extraction and stabilization of folates by derivatization. We perform reductive methylation employing stable isotope labeled reagents to retain information on the position and redox state of one-carbon units as well as the redox state of the pteridine ring. The derivatives are analyzed by a targeted LC(HILIC)-MS/MS method without the need for deconjugation, thereby also preserving the glutamation state of folates. The presented method does not only improve analyte coverage and sensitivity as compared to other published methods, it also greatly simplifies sample handling and storage. Finally, we report differences in the response of bacterial and mammalian systems to pharmacological inhibition of dihydrofolate reductase.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Unstable folate species and stabilized derivatives. (A) pH dependent equilibrium of folates carrying one-carbon units at the formic acid oxidation level. (B) General structure of the folate backbone. (C) Dimethylated folate without isotopic labels can be employed as an internal standard. (D–K) Derivatives of 5-formyl THF, 10-formyl THF, 5-methyl THF, 5,10-methylene THF, 5,10-methenyl THF, THF, DHF, and folic acid.
Figure 2
Figure 2
Method performance overview. (A) Concentrations of individual isotopologues are calculated by deconvolution. Crosstalk between adjacent channels caused by natural 13C isotopic abundances and imperfect labeling of reagents. Blue: Calculated values from theoretic isotopic distribution and specified labeling extent of reagents. Green: Experimental values from pure standards. (B) Stability of derivatized analytes over time at 4 °C. (C) Stability of derivatized folates during vacuum concentration. (D) Matrix effects of E. coli lysate (purple) and HepG2 lysate (green) at two different spiked in standard concentrations (conc) compared to buffer (blue). *p < 0.01.
Figure 3
Figure 3
Schematic of one-carbon cycle showing the connection between folate metabolism and methionine cycle (Scheme adapted from ref (21). Copyright Elsevier 2013). Color coding: purple, C1 donors; green, folates; blue, products of C1 metabolism and downstream pathways; white, not covered by method. Abbreviations: CTH, cystathionine γ-lyase; CBS, cystathionine β-synthase; DHF, dihydrofolate; FA, folic acid; Hcy, homocysteine; MAT, methionine adenosyltransferase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; 5-MTHF, 5-methyl-tetrahydrofolate; MT, methyltransferase; SAH, S-adenosylhomocysteine; SAHH, SAH hydrolase; SAM, S-adenosylmethionine; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; B6, vitamin B6; B12, vitamin B12.
Figure 4
Figure 4
MRM Chromatogram of derivatized folate extract from E. coli K12 grown on complex medium.
Figure 5
Figure 5
Effect of DHFR inhibition in bacterial and mammalian systems. (A) Distribution of polyglutamation in control and trimethoprim treated E. coli K12. Peak areas of all folate species with a given polyglutamation number were summed and normalized. (B) Relative changes (log 10-fold) of reduced folates upon trimethoprim treatment of E. coli K12. (C) Selected C1 targets in E. coli. (D) Distribution of polyglutamation in control and methotrexate treated HepG2 cells. (E) Relative changes (log 10-fold) of reduced folates upon methotrexate treatment of HepG2 cells. (F) Selected C1 targets in HepG2 cells. *p < 0.01.

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