Specific Method for the Determination of Genomic DNA Methylation by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry
- 4 December 2004
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 77 (2), 504-510
- https://doi.org/10.1021/ac0489420
Abstract
Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2‘-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2‘-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2‘-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2‘-deoxycytidine to 2‘-deoxyguanosine and was determined directly using 2‘-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2‘-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection.Keywords
This publication has 34 references indexed in Scilit:
- Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser‐ induced fluorescence detectionElectrophoresis, 2004
- DNA methylation in cancer: too much, but also too littleOncogene, 2002
- Reactivating the expression of methylation silenced genes in human cancerOncogene, 2002
- DNMT1 and DNMT3b cooperate to silence genes in human cancer cellsNature, 2002
- DNA of Drosophila melanogaster contains 5-methylcytosineThe EMBO Journal, 2000
- Liquid chromatographic determination of 5-methylcytosine in DNA with fluorescence detectionAnalytica Chimica Acta, 1998
- An Improved Sample Preparation Method for the Quantitative HPLC Determination of 5-Methyl-Deoxycytidine in Animal Tissue DNAJournal of Liquid Chromatography & Related Technologies, 1997
- Electrokinetic separations with micellar solutions and open-tubular capillariesAnalytical Chemistry, 1984
- Hypomethylation distinguishes genes of some human cancers from their normal counterpartsNature, 1983
- 5-Methylcytosine in Eukaryotic DNAScience, 1981