科研之友
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摘要
Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostaglandin, biosynthesis, can react with DNA to form stable adducts. These adducts may contribute to the development of such inflammation-mediated diseases as cancer and cardiovascular and neurodegenerative diseases. The predominant MDA-derived DNA adduct formed under physiological conditions is 3-(2-deoxy-beta-D-erythro-pentafuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one deoxyguanosine (M,(1)dG). In this study, we developed a novel liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high-resolution tandem mass spectrometry (HRMS/MS) method for the analysis of M,dG in human leukocyte DNA. After enzymatic hydrolysis of DNA, M(1)dG and the added internal standard [C-13(3)]M(1)dG were reduced to their 5,6-dihydro derivatives by addition of sodium borohydride to the hydrolysate and purified by solid-phase extraction and column chromatography. The 5,6-dihydro derivatives in the purified samples were analyzed by LC-NSI-HRMS/MS using higher-energy collisional dissociation (HCD) fragmentation, isolation widths of 1 Da for both the analyte and internal standard, and a resolution of 50000. The detection limit of the developed method is 5 amol on-column, and the limit of quantitation is 0.125 fmol/mg DNA starting with 200 pg of DNA. Method accuracy and precision were characterized. The developed method was further applied to the analysis of leukocyte DNA from 50 human subjects. M(1)dG was detected in all samples and ranged from 0.132 to 275 fmol/mg DNA, or 0.004 to 9.15 adducts per 108 bases. This unique and highly sensitive HRMS/MS-based method can be used in future studies investigating the pathophysiological role of M(1)dG in human diseases.
- 出版日期2014-10
