DNA Adduct Analysis via LC-MS

Cell pellets (1.5 – 2 × 10⁶) were homogenized in TE buffer (300 μL, 50 mM Tris-HCl containing 10 mM EDTA, and 10 mM BME, pH 8.0) and DNA was purified with the Gentra Puregene kit (Qiagen, Valencia, CA) (Cai et al. 2017 (link)). The DNA was washed with 70%, ethanol and reconstituted in LC/MS grade water. The concentration of DNA was determined using an Agilent 8453 UV/vis spectrophotometer (Agilent Technologies, Santa Clara, CA). Ten μg of DNA were spiked with isotopically labeled internal standards ([¹³C₁₀]-dG-C8–4-ABP, [¹³C₁₀]-dG-C8-PhIP, [¹³C₁₀]-dG-C8-AαC, [²H₃C]-dG-C8-MeIQx, each at a level of 1 adducts per 10⁷ nucleotides) and were digested with a cocktail of enzymes in 5 mM Bis-Tris-HCl buffer (pH 7.1) as previously described (Goodenough et al. 2007 (link); Nauwelaers et al. 2011 (link)). Isotopically labeled 2-NA DNA adducts were not available, and [¹³C₁₀]-dG-C8–4-ABP was used as a surrogate internal standard assuming similar ionization efficiencies for all adducts.