DNA methylation determination by liquid chromatography-tandem mass spectrometry using novel biosynthetic [U-15N]deoxycytidine and [U-15N]methyldeoxycytidine internal standards

Methylation of the promoter CpG regions regulates gene transcription by inhibiting transcription factor binding. Deoxycytidine methylation may regulate cell differentiation, while aberrations in the process may be involved in cancer etiology and the develop- ment of birth defects (e.g. neural tube defects). Similarly, nutritional deficiency and certain nutra- genomic interactions are associated with DNA hypo- methylation. While LC-MS has been used previously to measure percentage genomic deoxycytidine methylation, a lack of a secure source of internal standards and the need for laborious and time- consuming DNA digestion protocols constitute distinct limitations. Here we report a simple and inexpensive protocol for the biosynthesis of internal standards from readily available precursors. Using these biosynthetic stable-isotopic (U-15N)-labeled internal standards, coupled with an improved DNA digestion protocol developed in our lab, we have developed a low-cost, high-throughput (`500 sam- ples in 4 days) assay for measuring deoxycytidine methylation in genomic DNA. Inter- and intraassay variation for the assay (%RSD, n=6) was _2.5%.