Protocol for the simultaneous quantification of oxidative purine lesions in DNA using LC-MS/MS analysis

Most DNA damages induced through oxidative metabolism are single lesions which can accumulate in tissues. Here, we present a protocol for the simultaneous quantification of oxidative purine lesions (cPu and 8-oxo-Pu) in DNA. We describe steps for enzymatic digestion of DNA and sample pre-purification, followed by quantification through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We optimized this protocol in commercially available calf thymus DNA and used genomic and mitochondrial DNA extracted from cell cultures and animal and human tissues. [Display omitted] •Quantification of purine lesions in DNA from biological samples by LC-MS/MS•Synthesis of 15N-labeled 5′,8-cyclopurines and 8-oxo-purines for DNA lesions detection•Tailored enzymatic digestion of DNA for purine lesions detection•Oxidation-resistant procedure of sample cleanup, enrichment, and analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Most DNA damages induced through oxidative metabolism are single lesions which can accumulate in tissues. Here, we present a protocol for the simultaneous quantification of oxidative purine lesions (cPu and 8-oxo-Pu) in DNA. We describe steps for enzymatic digestion of DNA and sample pre-purification, followed by quantification through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We optimized this protocol in commercially available calf thymus DNA and used genomic and mitochondrial DNA extracted from cell cultures and animal and human tissues.