Identification and quantification of 8,5'-cyclo-2'-deoxy-adenosine in DNA by liquid chromatography/ mass spectrometry.
Recent studies suggested that 8,5'-cyclo-2'-deoxyadenosine may play a role in diseases with defective nucleotide-excision repair. This compound is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the sugar moiety of 2'-deoxyadenosine. It is likely to be repaired by nucleotide-excision repair rather than by base-excision repair because of a covalent bond between the sugar and base moieties. We studied the measurement of 8,5'-cyclo-2'-deoxyadenosine in DNA by liquid chromatography/isotope-dilution mass spectrometry. A methodology was developed for the analysis of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography in DNA hydrolyzed to nucleosides by a combination of four enzymes, i.e., DNase I, phosphodiesterases I and II, and alkaline phosphatase. Detection by mass spectrometry was performed using atmospheric pressure ionization-electrospray process in the positive ionization mode. Results showed that liquid chromatography/isotope-dilution mass spectrometry is well suited for identification and quantification of 8,5'-cyclo-2'-deoxyadenosine in DNA. Both (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyadenosine were detected. The level of sensitivity of liquid chromatography/mass spectrometry with selected-ion monitoring amounted to 2 fmol of this compound on the column. The yield of 8,5'-cyclo-2'-deoxyadenosine was measured in DNA in aqueous solution exposed to ionizing radiation at doses from 2.5 to 80 Gray. Gas chromatography/mass spectrometry was also used to measure this compound in DNA. Both techniques yielded similar results. The yield of 8,5'-cyclo-2'-deoxyadenosine was comparable to the yields of some of the other major modified bases in DNA, which were measured using gas chromatography/mass spectrometry. The measurement of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography/mass spectrometry may contribute to the understanding of its biological properties and its role in diseases with defective nucleotide-excision repair.
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899-8311, USA. miral@nist.gov
M
M
Dizdaroglu
P
P
Jaruga
H
H
Rodriguez
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004260:DNA Repair; D003839:Deoxyadenosines / Q000032:analysis*; D003850:Deoxyribonuclease I / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D013058:Mass Spectrometry; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D003839:Deoxyadenosines; C056752:8,5'-cyclo-2'-deoxyadenosine; D004247:DNA; D005092:Exonucleases; C022650:spleen exonuclease; D003850:Deoxyribonuclease I; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I