Mass spectrometric analysis of the active site tryptic peptide of recombinant O6-methylguanine-DNA methyltransferase following incubation with human colorectal DNA reveals the presence of an O6-alkylguanine adductome.

Rasha Abdelhady, Pattama Senthong, C.E. Eyers, Onrapak Reamtong, Elizabeth Cowley , Luca Cannizzaro, Joanna Stimpson, Kathleen Cain, Oliver Wilkinson , Nicholas Williams, Perdita Barran, G.P. Margison, David Williams, Andrew Povey

Research output: Contribution to journalArticlepeer-review

Abstract

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts were quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, temozolomide methylated calf thymus DNA (Me-CT-DNA) and human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin and ASPs detected and quantified by matrix assisted laser desorption/ionization-time of flight mass spectrometry. ASPs containing S-methyl, -ethyl, -propyl, -hydroxyethyl, -carboxymethyl, -benzyl, and -pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-Radioimmunoassay (r2 = 0.74; p=0.014). O6-CMG, a putative O6-hydroxyethylG adduct and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterised. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information on cancer pathogenesis.
Original languageEnglish
JournalChemical Research in Toxicology
Publication statusAccepted/In press - 1 Nov 2023

Keywords

  • O6 -alkylguanine
  • DNA adducts
  • MGMT
  • colorectal cancer
  • mass spectrometry

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