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

• DOI: 10.1021/acs.chemrestox.3c00207
• Publication Date: 2023-11-20T18:11:26Z
• Authors: Rasha Abdelhady; Pattama Senthong; Claire E. Eyers; Onrapak Reamtong; Elizabeth Cowley; Luca Cannizzaro; Joanna Stimpson; Kathleen Cain; Oliver J. Wilkinson; Nicholas H. Williams; Perdita E. Barran; Geoffrey P. Margison; David M. Williams; Andrew C. Povey
• Keywords: O-6-methylguanine-DNA methyltransferase; DNA methyltransferase

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