Quantitative determination of oxidative base damage in DNA by stable isotope‐dilution mass spectrometry

[摘要]

For understanding of the role of oxidative DNA damage in biological processes such as mutagenesis and carcinogenesis, it is essential to identify and quantify this type of DNA damage in cells. This can be achieved by gas chromatography/mass spectrometry. The present study describes the quantification of modified bases in DNA by isotope-dilution mass spectrometry with the use of stable isotope-labeled analogues as internal standards. A number of isotopically labeled DNA bases were synthesized. The mass spectra of their trimethylsilyl derivatives were recorded. Calibration plots were obtained for known quantities of modified bases and their isotope-labeled analogues. Quantification of various modified DNA bases by isotope-dilution mass spectrometry was demonstrated in isolated chromatin exposed to ionizing radiation. The results indicate that gas chromatography/stable isotope-dilution mass spectrometry is an ideally suited technique for selective and sensitive quantification of modified bases in DNA.

[学科分类] 生物化学/生物物理

[关键词] Free radical;Hydroxyl radical;Isotope-labeled analogue;Modified base;Oxidative DNA damage;Radiation damage;Selected-ion monitoring;O2 −;Superoxide radical;∗OH;hydroxyl radical;BSTFA;bis(trimethylsilyl)trifluoroacetamide;GC/MS-SIM;gas chromatogra - phy/mass spectrometry with selected-ion monitoring;rel.;relative;Gy;gray (J/kg);EI;electron-ionization;8-OH-Ade;8-hydroxyadenine;FapyGua;2;6-diamino-4-hydroxy-5-formamidopyrimidine;8-OH-Gua;8-hydroxyguanine.