Hypersensitive quantification of global 5-hydroxymethylcytosine by chemoenzymatic tagging

5-hydroxymethylcytosine (5hmC) is an epigenetic DNA modification. Tissue-specific reduction in global 5hmC levels has been associated with various types of cancer. One of the challenges associated with detecting 5hmC levels is its extremely low content, especially in blood. The gold-standard for reliable global 5hmC quantitation is liquid chromatography-tandem mass spectroscopy (LC-MS/MS) operating in a multiple reaction monitoring (MRM) mode. Difficulties associated with 5hmC detection by LC-MS/MS include its low abundance, low ionization efficiency and possible ion suppression from co-eluted compounds. Hence, detecting 5hmC levels in blood samples for diagnosis of leukemia and other blood malignancies presents a unique challenge. To overcome these difficulties we introduce a simple chemoenzymatic method for specifically tagging 5hmC, resulting in an eight-fold increase in detection sensitivity. We demonstrate that we could quantitatively detect 5hmC levels in various human tissues, including blood samples from healthy individuals and leukemia patients, using the most basic quadrupole mass-analyzer instrument and only 200 ng of DNA. The limit of detection (LOD) of our technique is 0.001% 5hmC from 300 ng DNA, sufficient for future mass-spectroscopy based diagnostics of diseases associated with low 5hmC levels such as leukemia.