Materials and Reagents

1. NH₄HCO₃ (reagent grade ≥ 99% purity)
   
2. MgCl₂ (reagent grade ≥ 99% purity)
   
3. CaCl₂ (reagent grade ≥ 99% purity)
   
4. DNase I from bovine pancreas (F. Hoffmann-La Roche, catalog number: 04716728001 )
   
5. Nuclease P1 from P. citrinum (Sigma-Aldrich, catalog number: N8630-1VL )
   
6. Phosphodiesterase I from C. adamanteus venom (Sigma-Aldrich, catalog number: P3242-1VL )
   
7. Alkaline phosphatase from E. coli (Sigma-Aldrich, catalog number: P5931-100UN )
   
8. 8-hydroxy-2’-deoxyguanosine (8-oxo-dGuo) (Sigma-Aldrich, catalog number: H5653-1MG )
   
9. [¹⁵N₅]-8-hydroxy-2’-deoxyguanosine (Cambridge Isotope Laboratories, catalog number: NLM-6715-0 ) (this is the internal standard)
   
10. DNeasy^® Blood & Tissue Kit (QIAGEN, catalog number: 69506 )
    
11. LC/MS-grade methanol
    
12. Hydrolysis buffer (see Recipes)
    
13. Solvent A (see Recipes)
    
14. Solvent B (see Recipes)
    

Equipment

1. Vortexer
   
2. Microcentrifuge
   
3. Vacuum centrifuge
   
4. LC/MS/MS: We used an LC-20AD HPLC system (Shimadzu Corporation) coupled to an API 5000 triple-quadrupole mass spectrometer (Applied Biosystems)
   
5. Zorbax SB-C18 reverse phase chromatography column (2.1 x 150 mm, i.d., 3.5 μm) (Agilent Technologies)
   

Procedure