Eight units of nuclease P1, 0.01 unit of phosphodiesterase 2, 20 nmol of EHNA and a 20-μL solution containing 300 mM sodium acetate (pH 5.6) and 10 mM zinc chloride were added to 80 μg of DNA. In this context, some enzymes used for DNA digestion were contaminated with adenine deaminase, which can induce the deamination of dA to 2′-deoxyinosine (dI). Because dI shared a very similar retention time as S-cdG during HPLC enrichment (Figure S3), the presence of considerable amount of dI in the digestion mixture diminished substantially the sensitivity for S-cdG measurement. Therefore, we added EHNA prior to the enzymatic digestion 40 (link), which reduced the deamination of dA to less than 2%, thereby rendering S-cdG to be detected with adequate sensitivity. The above digestion was continued at 37°C for 48 h. To the digestion mixture were then added 8 units of alkaline phosphatase, 0.02 unit of phosphodiesterase 1 and 40 μL of 0.5 M Tris-HCl buffer (pH 8.9). The digestion mixture was incubated at 37°C for 2 h and subsequently neutralized by addition of formic acid. To the mixture were then added isotopically labeled standard lesions, which included 2.5 pmol of 5-FodU, 1.5 pmol of 5-HmdU, 80 fmol of R-cdG, 40 fmol of S-cdG, 60 fmol of R-cdA and 20 fmol of S-cdA (Scheme 1). The enzymes in the digestion mixture were subsequently removed by chloroform extraction. The resulting aqueous layer was dried, reconstituted in doubly distilled water and subjected to off-line HPLC separation for the enrichment of the lesions under study.