Ultraperformance Liquid Chromatography Tandem Mass Spectrometry Assay of DNA Cytosine Methylation Excretion from...

Measuring DNA cytosine methylation excretion presents challenges because methylated cytosine species are released in various forms including free molecules and those bound in DNA fragments. Herein, we report a novel UPLC-MS/MS method that allows the quantification of both free and DNA fragment-bound forms of methylated cytosine species excreted, providing total amounts for each. Cell culture medium and genomic DNA isolated from cells are analyzed to quantify methylated cytosine species. In genomic DNA isolated from MDA-MB-231 breast cancer cells, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are detected at 5.1% and 0.07% of total cytosine residues, respectively. In the cell culture medium, only 5hmC is detected at a low level (ca. 7 nM). However, in two normal cell lines (i.e., primary mouse lung epithelial cells and HEK293 kidney cells) 5mC, 5-methylcytidine, and 2'-oxymethylcytidine (but no 5hmC) are found present in cell culture medium at concentrations ranging from 10 to 320 nM. Further, it is observed for the first time that treating MDA-MB-231 cells with carboplatin significantly increases the 5hmC level in the culture medium, indicating a carboplatin-boosted DNA cytosine methylation excretion from cancer cells.