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Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation, sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column.

ABSTRACT: In this manuscript, an efficient high resolution reversed phase-ion pairing-liquid chromatography electrospray ionization tandem mass spectrometry (RP-IP-LC-MS/MS) method for separation of isomeric modified oligonucleotides using a polymeric (styrene-divinylbenzene) monolithic capillary column is presented. The effects of different ion pairing reagents (IPR), their concentration, mobile phase additives and conditions were evaluated towards achieving the highest possible resolution and chromatographic separation of isomeric oligonucleotides. Ion pairing reagents and mobile phase conditions were evaluated using as model N-acetylaminofluorene [AAF] adducted ss-oligonucleotides (CCC CGA GCA ATC TCA AT). The optimized mobile phase conditions were then applied for the mapping of sites of base modification of AAF adducted 15-base pair oligonucleotide fragments containing codon 135 of the p53 gene and for profiling a complex synthetic oligonucleotide mixture. The optimized method utilizes a monolithic poly(styrene-divinylbenzene) capillary column, triethylammonium bicarbonate as ion pairing reagent and methanol as organic modifier to perform IP-RPLC-ESI-MS/MS separation. The results show that the method is simultaneously applicable not only to oligonucleotide fragments adducted separately by different carcinogens but also to the analysis of multiple adducts in the same oligonucleotide fragment in a single experiment. The method presents itself as a tool for the identification, characterization and mapping of oligonucleotide adducts as biomarkers for DNA damage from carcinogens.

SUBMITTER: Sharma VK

PROVIDER: S-EPMC3372605 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation, sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column.

Sharma Vaneet K VK Glick James J Vouros Paul P

Journal of chromatography. A 20120509

In this manuscript, an efficient high resolution reversed phase-ion pairing-liquid chromatography electrospray ionization tandem mass spectrometry (RP-IP-LC-MS/MS) method for separation of isomeric modified oligonucleotides using a polymeric (styrene-divinylbenzene) monolithic capillary column is presented. The effects of different ion pairing reagents (IPR), their concentration, mobile phase additives and conditions were evaluated towards achieving the highest possible resolution and chromatogr ...[more]

PMID: 22652552

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Project description:The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce pyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN and NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitative analysis of four POB-DNA adducts with known structures. The corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis. The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis. The method was accurate and precise. Excellent sensitivity was achieved, especially for O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 12) was the most abundant adduct, followed by O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 8), O2-POB-dThd, and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O2-POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the in vitro data, 7-POB-Gua was the major POB-DNA adduct formed in vivo. However, levels of O6-POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in vivo. In contrast to the other three adducts, O6-POB-dGuo was more abundant in lung than in liver. O2-POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive quantitation of four POB-DNA adducts, support an important role of O6-POB-dGuo in NNK lung tumorigenicity in rats, and suggest that O2-POB-dThd may be a useful tobacco-specific DNA biomarker for future tobacco carcinogenesis studies.

Dataset Information

Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation, sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column.

Publications

Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation, sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column.

Similar Datasets

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