Analysis of DNA-phosphate adducts in vitro using miniaturized LC-ESI-MS/MS and column switching: Phosphotriesters and alkyl cobalamins

Abstract

Belgium DNA-phosphate adducts are known to be formed by a variety of alkylating agents. Due to little or no repair of DNA-phosphate adducts, these adducts may offer increased possibilities of both identifying and quantifying DNA adducts. The formation of DNA-phosphate adducts leads to a complete esterification of the phosphate group giving rise to a phosphotriester configuration. This work consists of the characterization of ethyl phosphotriesters (Ethyl PTE) using miniaturized LC-ESI-MS/MS and column switching in enzymatic hydrolysate of DNA treated in vitro with the model compound N-ethyl-N-nitrosourea (ENU). In vitro ENU-treated DNA was enzymatically degraded using nuclease P1, phosphodiesterase, and alkaline phosphatase. The use of column switch allowed for large-volume injections, where unmodified nucleosides were discarded in the loading step. The analytes were forward flushed to the analytical column in the eluting step and separated using a linear gradient. Ten different ethyl PTEs (dGpEtdG, dApEtdA, dCpEtdC, TpEtT, dGpEtdA, dGpEtdC, dGpEtT, dApEtdC, dApEtT, and dCpEtT) were characterized by their masses and CAD product ion spectra. Measurements of accurate masses were carried out yielding experimental masses within 5 ppm of the calculated masses for 9 of the 10 ethyl PTEs. For comparison, the enzymatic hydrolysate of ENU-treated DNA was subjected to transalkylation of the DNA-phosphate adducts by cob(I)alamin. Formed ethyl-cobalamins were analyzed according to earlier developed methods. The limit of detection of an alkyl-cobalamin standard and an alkyl PTE standard was 2 fmol and 5 fmol, respectively. (J Am Soc Mass Spectrom 2004, 15, 593-606)

Adduct levels from benzo[a]pyrenediol epoxide: Relative formation to histidine in serum albumin and to deoxyguanosine in DNA in vitro and in vivo in mice measured by LC/MS–MS methods

“…To obtain a qualitative standard for method development and MS analysis, DNA was alkylated in vitro with BPDE generally as described before (Barry et al, 1996). The incubation was conducted for 3 h at 37 C. A pellet containing the BPDE-alkylated DNA was carefully dried before enzymatic hydrolysis with endonuclease nuclease P1 (NP1, 80 units/mg of DNA) and exonuclease snake venom phosphodiesterase I (0.08 units/mg of DNA) as previously described (Crain 1990;Haglund et al, 2004). Dephosphorylation of the adducts to deoxyribonucleosides (dN) was obtained by the addition of alkaline phosphatase (20 units/mg of DNA) to the sample and incubation at 37 C for 1.5 h. The sample was centrifuged at 20,000 Â g and the supernatant was thereafter ready for LC/MS-MS analysis.…”

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

“…A column switching approach was used to remove the unmodified 2 0 -deoxynucleosides following a further digestion of the DNA sample by incubation with a 5 0 -phosphodiesterase and alkaline phosphatase. Ten different ethyl phosphotriester dinucleotide adducts were characterized by their CID product ion spectra (148).…”

Simultaneous determination of O6-methyl-2′-deoxyguanosine, 8-oxo-7,8-dihydro-2′-deoxyguanosine, and 1,N6-etheno-2′-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

“…The effectiveness of column switching methods for the analysis of adducts was recently confirmed for DNA in vitro [10,18], nucleosides in urine [19] and for DNA in tissue [20,21]. Therefore, a HPLC-MS/MS method using an on-line column switching technique was developed and validated for the simultaneous analysis of the promutagenic DNA adducts O 6 -mdGuo, 8-oxodGuo, and dAdo.…”