Glycosylase Pretreatment with Chemical Labeling-Assisted HPLC-MS/MS: An Ultrasensitive and Reliable Strategy for Quantification of 8-Oxo-7,8-dihydro-2′-deoxyguanosine in Genomic DNA

Abstract

8-Oxo-7,8-dihydro-2′-deoxyguanosine (dOG), the dominant oxidative product of 2′-deoxyguanosine (dG) under high levels of reactive oxygen species, usually serves as a biomarker for oxidative stress and a risk assessment factor for various diseases. Due to the extremely low abundance of dOG and the susceptibility of dOG detection to the interference of spurious oxidation, research on related biological processes is limited by insufficient sensitivity and specificity. In this work, an ultrasensitive and reliable approach for genome-wide dOG quantification was developed through chemical labeling-assisted high-performance liquid chromatography-tandem mass spectrometry with the introduction of glycosylase pretreatment. Upon derivatization by a novel labeling reagent rhodamine B ethylenediamine, the detection sensitivity of dOG was enhanced by 100-fold, and the detection limit was as low as 25 amol, which was superior to those of reported mass spectrometry-based methods. Potassium ferricyanide, as a single-electron oxidant, was shown to possess strong selectivity for dOG versus dG, improving the labeling specificity and reducing the interference from dG. The spurious oxidation during sample pretreatment was systematically explored and minimized, and a control assay of glycosylase pretreatment was proposed to further improve the quantitative accuracy of dOG. Precise quantification of endogenous dOG in different cells was achieved with less than 500 ng of genomic DNA. This method was successfully applied to the assessment of the overall level of oxidative damage under the treatment of glycosylase inhibitors, potentially contributing to the exploration of the complex role of dOG in physiological status and disease phenotype.