title: Analysis of a malondialdehyde-deoxyguanosine adduct in human leukocyte DNA by liquid chromatography nanoelectrospray-high-resolution tandem mass spectrometry.
abstract: 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-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG). 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 M1dG in human leukocyte DNA. After enzymatic hydrolysis of DNA, M1dG and the added internal standard [(13)C3]M1dG 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 50 000. 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 μg of DNA. Method accuracy and precision were characterized. The developed method was further applied to the analysis of leukocyte DNA from 50 human subjects. M1dG was detected in all samples and ranged from 0.132 to 275 fmol/mg DNA, or 0.004 to 9.15 adducts per 10(8) bases. This unique and highly sensitive HRMS/MS-based method can be used in future studies investigating the pathophysiological role of M1dG in human diseases.
authors.affiliation: Masonic Cancer Center and ‡Division of Environmental Health Sciences, University of Minnesota , Mayo Mail Code 806, 420 Delaware Street South East, Minneapolis, Minnesota 55455, United States.
authors.forename: Bin
authors.initials: B
authors.lastname: Ma
authors.forename: Peter W
authors.initials: PW
authors.lastname: Villalta
authors.forename: Silvia
authors.initials: S
authors.lastname: Balbo
authors.forename: Irina
authors.initials: I
authors.lastname: Stepanov
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis* / Q000302:isolation & purification; D003849:Deoxyguanosine / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008315:Malondialdehyde / Q000737:chemistry*; D036103:Nanotechnology; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry
chemical_list: D018736:DNA Adducts; D008315:Malondialdehyde; D004247:DNA; D003849:Deoxyguanosine