Nucleic Acids Research Advance Access published online on July 18, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm526
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Chemistry |
Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry
Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, UK
*To whom correspondence should be addressed: Tel: +44 0 116 2231827; Fax: +44 0 116 2231840; Email: rs25{at}le.ac.uk
Received May 24, 2007. Accepted June 22, 2007.
In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2'-deoxynucleoside 3'-monophosphates. The B[a]PDE plus 2'-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M–H]– consistent with B[a]PDE adducted (either on the base or phosphate group) 2'-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)–H]–, resulting from cleavage of the glycosidic bond between the 2'-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2'-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2'-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)–H]– and [(2'-deoxyribose+phosphate+B[a]Ptriol)–H]–, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2'-deoxynucleotides.
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