Mass Spectroscopy for Detecting Epigenetic Variations in Human Blood Cancer

Epigenetic modifications include dynamic chemical alterations on specific DNA bases that do not involve changes in the DNA sequence. In particular, 5-hydroxymethylcytosine (5-hmC) is a tissue-type specific epigenetic modification on the base cytosine. Reduction in 5-hmC levels has been recently associated with various types of cancers, such as leukemia, indicating its potential as a biomarker for early disease diagnosis. LC-MS/MS is the most reliable technique for 5-hmC detection. However, detecting 5-hmC levels is extremely challenging, especially in blood which contains very low 5hmC content, due to its low ionization efficiency and interference from other nucleobases. Here we introduce a simple enzymatic procedure for selective labeling of 5-hmC with a molecule of high ionization efficiency, resulting in eight folds increase in the LC-MS/MS response to 5-hmC. The limit of quantification (LOQ) of our technique is 0.001% 5-hmC per total nucleotides (below physiologically relevant levels), using only 500 ng DNA per measurement. We demonstrate that we can quantitatively differentiate between 5-hmC levels in healthy and leukemia blood samples, using just 200 ng DNA per measurement. Thus, our method may open a new era of mass spectroscopy-based diagnostics for early detection of diseases associated with extremely reduced 5-hmC levels, such as leukemia.