Quantification of DNA Modifications Using Two-Dimensional Ultraperformance Liquid Chromatography Tandem Mass Spectrometry (2D-UPLC-MS/MS).

Our hereby presented methodology is suitable for reliable assessment of the most common DNA modifications which arise as a product of fundamental metabolic processes. 8-oxoguanine, one of the oxidatively modified DNA bases is a typical biomarker of oxidative stress. A noncanonical base, uracil, may also be present in small quantities in DNA. Ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can also be generated by TET enzymes. All the above mentioned modifications seem to play some regulatory roles. Here, we provide a protocol for isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. We also provide optimized protocols for extraction of DNA, fully compatible with the downstream MS/MS analysis.