Analysis of glyoxal-induced DNA cross-links by capillary liquid chromatography nanospray ionization tandem mass spectrometry.
Glyoxal (gx) is an alpha-dicarbonyl species derived endogenously from the metabolism of carbohydrates or nitrosamines and from oxidation of lipids and nucleic acids. It is also widely distributed in foods and the environment. Glyoxal reacts with biomolecules, causing cross-links of proteins and DNA. The cross-linked products of glyoxal with 2'-deoxyribonucleosides have been characterized as dG-gx-dC, dG-gx-dG, and dG-gx-dA. We herein develop a highly specific and sensitive capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) assay for the simultaneous quantification of these three DNA cross-links using a triple-quadrupole mass spectrometer. The sample pretreatment procedures included enzyme hydrolysis of DNA and adduct enrichment by a reversed phase solid phase extraction column. We compared two enzyme hydrolysis conditions, and significantly different adduct levels were observed. This assay achieved attomole sensitivity with detection limits of 12-75 amol injecting each cross-link standard on-column. After calf thymus DNA was incubated with 1.0 mM of glyoxal at 37 degrees C for 30 days, the levels of dG-gx-dC, dG-gx-dG, and dG-gx-dA in this sample were determined as 6.52, 0.80, and 2.74 in 10(5) normal nucleotides, respectively, by capLC-NSI/MS/MS analysis after hydrolysis under optimized conditions. The identity of these cross-links in glyoxal-treated DNA was confirmed by MS(2) and MS(3) scan spectra using a linear ion trap mass spectrometer. In 20 microg of human placental DNA hydrolysate, the levels of dG-gx-dC, dG-gx-dG, and dG-gx-dA were quantified as 2.49, 1.26, and 3.50 in 10(8) normal nucleotides, respectively. These DNA cross-links, if not repaired, can be mutagenic, and they represent a type of damage to the integrity of DNA structure due to exposure of glyoxal.
Chen
Hauh-Jyun Candy
HJ
Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan. chehjc@ccu.edu.tw
Chen
Yu-Chin
YC
D002853:Chromatography, Liquid / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis*; D004249:DNA Damage; D006037:Glyoxal / Q000737:chemistry*; D006801:Humans; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D006037:Glyoxal