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.
Highlights
One-carbon (C1) metabolism is a central metabolic pathway which distributes C1 units derived from C1 donors to several crucial cellular pathways, namely, purine synthesis,[1] thymidine synthesis,[2] and the S-adenosyl methionine cycle.[3]
The pteridine ring of C1 carrying folates is typically fully reduced to tetrahydrofolate (THF, Figure 1B), and C1 units are either attached to nitrogen 5 (N5), nitrogen 10 (N10), or both forming a bridge between the nitrogen moieties
To prevent interconversion of the three species, the free C1 binding site has to be chemically blocked. Both N5 and N10 are secondary amines, and the choice of potential derivatization reactions is limited by the aqueous environment
Summary
One-carbon (C1) metabolism is a central metabolic pathway which distributes C1 units derived from C1 donors to several crucial cellular pathways, namely, purine synthesis,[1] thymidine synthesis,[2] and the S-adenosyl methionine cycle.[3]. Since superior sensitivity of folate anaylsis in positive mode was removed, and cells were immediately resuspended in 0.5 mL of reported before,[14] we tested two HILIC phases, namely, the ice cold 80% methanol, containing 30 mM NaCNBD3, 0.2% formaldehyde-13C, d2, and 0.1% acetic acid by pipetting.
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