Analysis of benzo[a]pyrene diol epoxide‐DNA adducts by capillary zone electrophoresis‐ electrospray ionization‐mass spectrometry in conjunction with sample stacking

Abstract

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.

Ultra-performance liquid chromatography–tandem mass spectrometry for rapid and highly sensitive analysis of stereoisomers of benzo[a]pyrene diol epoxide–DNA adducts

“…Benzo[a]pyrene (B[a]P) is a representative of carcinogenic PAH. It is metabolized in vivo to an ultimate carcinogen, anti-benzo [a]pyrene diol epoxide (BPDE), which may bind to an exocylic amine group at N 2 -deoxyguanosine (dG) or N 6 -deoxyadenosine(dA) in DNA [8]. It is possible to form four stereoisomers of anti-BPDE-N 2 dG and four stereoisomers of anti-BPDE-N 6 dA, derived from the reaction of (±)-anti-BPDE with DNA [9].…”

Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection

“…Using CZE-MS to analyse 2 0 -deoxynucleoside 5 0 -monophophates, Willems et al identified the formation of adducts with exocyclic -NH 2 groups of guanine, adenine and cytosine following the reaction of benzo[a]pyrene diol epoxide with calf thymus DNA. The authors used an online sample stacking technique for the pre-concentration of the negatively charged adducts and removal of sample buffer (196). A similar approach was used by Barry et al to analyse enzymatically hydrolysed calf thymus DNA treated with benzo[a]pyrene diol epoxide for which the authors noted the presence of an adducted thymidine-guanine dinucleotide in addition to a 2 0 -deoxyguanosine 5 0 -monophosphate adduct of benzo[a]pyrene diol epoxide (197).…”

Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis‐electrospray ionization‐quadrupole time of flight‐mass spectrometry

“…Because of the nature of the stacking process, the method lends itself particularly well to the analysis of negatively charged species and, as a consequence, to the analysis of oligonucleotides [34,36,37,40]. This sample stacking technique has already been used with success by our research group and the groups of Vouros et al for the analysis of deoxynucleotide DNA adducts of phenyl glycidyl ethers [40] and benzo[a]pyrene [37,41] and the analysis of oligonucleotides [10] by CZE coupled to ESI-MS.…”

“…Sample stacking was necessary to preconcentrate the oligonucleotide samples and was performed as described earlier with minor modifications [41]. The voltage of reversed polarity was tested in the range of 27 to 230 kV, resulting in the best signal response when 220 kV was used.…”