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

Abstract

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.

Figure 1.

HPLC-fluorescence chromatogram of a reaction mixture containing (±)-anti-B[a]PDE (250 μg) and 0.1 M TRIS base pH 7.0 buffer incubated at 37°C for 18 h and subjected to solid phase extraction. The analysis was performed using gradient elution with 0.05 M potassium phosphate buffer, pH 7.2 (solvent A) and acetonitrile (solvent B) at a flow of 1 ml/min (* = refer to Figures 2A, 4A and 5A).

Figure 2.

HPLC-fluorescence chromatogram of the reaction of B[a]PDE (250 μg) with dGp (1 mg) following gradient elution with 0.05 M potassium phosphate buffer, pH 7.2 (solvent A) and acetonitrile (solvent B) at a flow of 1 ml/min (A) Typical negative ESI LC-MS/MS CID product ion spectra for fractions 1 and 2 corresponding to phosphodiester adducts (B) and fraction 3 corresponding to base adduct (C). Both spectra were obtained from the molecular ion [M−H]⁻ at m/z 648 following isocratic elution with methanol/HPLC grade water (45:65, v/v) at a flow rate of 120 μl/min and collision energy of 21 eV [* = peaks present in control reaction mixture (Figure 1)].

Figure 3.

HPLC-fluorescence chromatogram of the reaction of B[a]PDE (250 μg) with dAp (1 mg) following gradient elution with 0.05 M potassium phosphate buffer, pH 7.2 (solvent A) and acetonitrile (solvent B) at a flow of 1 ml/min (A). Typical negative ESI LC-MS/MS CID product ion spectra for fractions 1, 2 and 3 corresponding to phosphodiester adducts (B) fraction 4 corresponding to base adduct (C). Both spectra were obtained from the molecular ion [M−H]⁻ at m/z 632 following isocratic elution with methanol/HPLC grade water (45:65, v/v) at a flow rate of 120 μl/min and collision energy of 21 eV.

Figure 4.

HPLC-fluorescence chromatogram of the reaction of B[a]PDE (250 μg) with dCp (1 mg) following gradient elution with 0.05 M potassium phosphate buffer, pH 7.2 (solvent A) and acetonitrile (solvent B) at a flow of 1 ml/min (A). Typical negative ESI LC-MS/MS CID product ion spectra for fractions 1, 2 and 3 corresponding to phosphodiester adducts (B) fraction 4 corresponding to base adduct. (C) Both spectra were obtained from the molecular ion [M−H]⁻ at m/z 608 following isocratic elution with methanol/HPLC grade water (45:65, v/v) at a flow rate of 120 μl/min and collision energy of 21 eV [* = peaks present in control reaction mixture (Figure 1)].

Figure 5.

HPLC-fluorescence chromatogram for the reaction of B[a]PDE (250 μg) with Tp (1 mg) following gradient elution with 0.05 M potassium phosphate buffer, pH 7.2 (solvent A) and acetonitrile (solvent B) at a flow of 1 ml/min (A). Typical negative ESI LC-MS/MS CID product ion spectrum for fractions 1 and 2 corresponding to phosphodiester adducts (B). The spectrum was obtained from the molecular ion [M−H]⁻ at m/z 623 following isocratic elution with methanol/HPLC grade water (45:65, v/v) at a flow rate of 120 μl/min and collision energy of 21 eV [* = peaks present in control reaction mixture (Figure 1)].

Figure 6.

Typical positive ESI LC-MS/MS CID product ion spectra for the base adducted 2′-deoxynucleotides. The spectra were obtained from the molecular ions [M+H]⁺ at m/z 650, B[a]PDE plus dGp (fraction 3, Figure 2A) (A), m/z 634 B[a]PDE plus dAp [fraction 4 (×2 concentrated), Figure 3A] (B) and m/z 610 B[a]PDE plus dCp [fraction 4 (×2 concentrated), Figure 4A] (C) following isocratic elution with 0.1% acetic acid/methanol (50:50, v/v) at a flow rate of 120 μl/min. The MS conditions used are as described in the Experimental Procedures section except for the following; capillary voltage, 3.20 kV and collision energy, 16 eV.

Scheme 1.

Reaction of B[a]PDE with the sugar phosphate backbone of DNA and postulated mechanism for strand scission [adapted from Gamper et al. (29)].

Scheme 2.

Negative ESI-MS/MS CID fragmentation pathway for B[a]PDE 2′-deoxynucleotide phosphodiester adducts.

Scheme 3.

Negative ESI-MS/MS CID fragmentation pathway for B[a]PDE base 2′-deoxynucleotides adducts (ions in brackets were not observed).