Use of 13C315N1-Serine or 13C515N1-Methionine for Studying Methylation Dynamics in Cancer Cell Metabolism and Epigenetics
Tracing the fate of carbon-13 (13C) labeled metabolites within cells by liquid chromatography mass spectrometry (LCMS) is a powerful analytical technique used for many years in the study of cell metabolism. Conventional experiments using LCMS and labeled nutrients tend to track the incorporation of 13C from exogenous nutrients (such as amino acids) into other, relatively proximal, cellular metabolites. Several labs have extended this technique to track transfer of 13C from the metabolite pool onto macromolecules, such as DNA, where methylation acts as an important functional modification. Here we describe a complete method that integrates previously established techniques to simultaneously track the use of 13C-serine or 13C-methionine into metabolite pools of the methionine cycle and into methylation of DNA and RNA. Given the ability to track methyl-transfer in a time-dependent way, this technique can provide temporal information about active methyl-transfer as well as quantification of total DNA/RNA methylation levels.
Key wordsMethylation Methyl-transfer DNA RNA Flux Carbon-13 One-carbon metabolism Methionine Serine Liquid chromatography mass spectrometry
ODKM and TZ are funded by Cancer Research UK Career Development Fellowship C53309/A19702. KHV and CFL are funded by CRUK Grant C596/A10419.
- 5.Herbig K, Chiang EP, Lee LR, Hills J, Shane B, Stover PJ (2002) Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses. J Biol Chem 277(41):38381–38389. https://doi.org/10.1074/jbc.M205000200CrossRefPubMedGoogle Scholar
- 6.Kok RM, Smith DE, Barto R, Spijkerman AM, Teerlink T, Gellekink HJ, Jakobs C, Smulders YM (2007) Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects. Clin Chem Lab Med 45(7):903–911. https://doi.org/10.1515/CCLM.2007.137CrossRefPubMedGoogle Scholar