Specific Method for the Determination of Genomic DNA Methylation by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

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.

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

“…For this task, we utilized a genetic DNMT knockout HCT116 model system (Rhee et al, 2000(Rhee et al, , 2002. We initially confirmed the genotype and phenotype of the DNMT knockout lines by measuring DNMT1 and DNMT3b expression by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively (Supplemental Figure 2a and b), and by analysing global genomic DNA methylation levels using Liquid chromatography-mass spectrometry (LC-MS) (Song et al, 2005) (Supplemental Figure 2c). The LC-MS assay revealed that genomic 5mdC levels are moderately depressed (B20%) in DNMT1À/À cells, slightly reduced in DNMT3bÀ/À cells (B2%), and greatly reduced (B70%) in double knockout cells (DKO) (Supplemental Figure 2c), indicating that the two enzymes cooperate to maintain genomic DNA methylation (Rhee et al, 2002).…”

“…Wild-type, DNMT1À/À, DNMT3bÀ/À, and DNMT1/3b double knockout (DKO) HCT116 colorectal cancer cell lines were a generous gift from Dr Bert Vogelstein (Johns Hopkins University School of Medicine), and were cultured as described previously (Song et al, 2005). The colon adenocar-cinoma cell line RKO was obtained from the ATCC (Rockville, MD, USA), and was cultured as described previously (Karpf et al, 2001).…”

“…Global genomic DNA methylation analyses Genomic DNA samples were isolated using the Puregene kit (Gentra Systems, Minneapolis, MN, USA) as described previously (Song et al, 2005). Genomic DNA methylation was assessed by direct measurement of the level of 5-methyl-2 0 -deoxycytidine (5mdC) using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS), as described previously (Song et al, 2005).…”

“…Genomic DNA methylation was assessed by direct measurement of the level of 5-methyl-2 0 -deoxycytidine (5mdC) using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS), as described previously (Song et al, 2005). Briefly, 1 mg genomic DNA samples were enzymatically hydrolysed with Nuclease P1 or Nuclease S1 into component nucleosides, and loaded, along with standards, onto an LC-ESI/MS/MS system.…”

Analysis of DNA modifications in aging research

“…Samples were then compared to a set of standards with known methylation levels (Reddy and Reddy 1990;Fuke et al 2004). Updates to this method include replacing UV detection with mass spectrometry (Song et al 2005). For more simple assays, enzyme-linked immunosorbent assays (ELISA) use antibodies that are specific for mC or hmC with an in vitro generated standard curve to measure the global levels of methylation in a given sample (Kalani et al 2014).…”