Glycosylase\nPretreatment with Chemical Labeling-Assisted\nHPLC-MS/MS: An Ultrasensitive and Reliable Strategy for Quantification\nof 8‑Oxo-7,8-dihydro-2′-deoxyguanosine in Genomic DNA

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