Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O 6 -Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O 6 -Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O 6 -methylguanine O 6 -methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O 6 -alkylguanines ( O 6 -alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O 6 -alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O 6 -alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O 6 -MethylG ( O 6 -MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S -methyl, S -ethyl, S -propyl, S -hydroxyethyl, S -carboxymethyl, S -benzyl, and S -pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O 6 -alkGs. The LOQ of ASPs containing S -methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O 6 -MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S -methylcysteine at levels that correlated with those of O 6 -MeG determined previously by HPLC-radioimmunoassay ( r 2 = 0.74; p = 0.014). O 6 -CMG, a putative O 6 -hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O 6 -alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O 6 -alkG adductome and, given the mutagenic potential of O 6 -alkGs, can provide mechanistic information about cancer pathogenesis.