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. [Display omitted] •Specific chemoenzymatic tagging of 5hmC results in eight folds signal enhancement in LC-MS/MS measurements.•A detection limit of 0.001% 5hmC from only 300 ng (9 fmol) DNA achieved with a basic LC-MS/MS device.•A reduction in %5hmC in leukemia patients was detected relative to healthy individuals, from only 200 ng DNA.•Proven reproducibility and accuracy of %5hmC levels in blood, solid tissues and synthetic DNA samples.•A potential for routine 5hmC% detection in clinical laboratories, using cost-effective LC/MS-MS devices.