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

Abstract

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, 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), or human colorectal DNA of known O⁶-MethylG (O⁶-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 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 characterized. 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 about cancer pathogenesis.

Figure 1

MALDI-ToF mass spectra of tryptic peptides of MBP-MGMT following its incubation with double-stranded 23-mer ODNs containing (A) O⁶-MeG, (B)O⁶-CMG, or (C) guanine. 2 pmol of active MBP-MGMT was incubated for 6 h at 37 °C with 20 pmol of 5′-GAA CTY CAG CTC CGT GCT GGC CC where Y is (A) O⁶-MeG, (B) O⁶-CMG, or (C) G and then digested with trypsin. Peak intensities are shown in arbitrary units on the y-axis, and the intensity scale is the same for all panels. The tryptic peptides detected included unmodified MGMT-ASP (GNPVPILIPCHR, 136–147) m/z 1315.73, methylated MGMT-ASP (GNPVPILIP(Me-C)HR, 136–147) m/z 329.74, and carboxymethylated MGMT-ASP (GNPVPILIP(CM-C)HR, 136–147) m/z 1373.73. The peak at m/z 1336.544 is an MBP peptide (SYEEELAKDPR, 332–342).

Figure 2

MALDI-ToF mass spectra analysis of alkylated ASPs present after incubating his-MGMT with human DNA. His-MGMT (50 pmol) was incubated with 2 mg of human colorectal DNA samples (A) 96T, (B) 25T, or (C) unmodified CT-DNA. Peak intensities are shown in arbitrary units on the y-axis, and the intensity scale is the same for all panels. S/N > 10 for all detected alkylated MGMT ASPs. The tryptic peptides (residues 136–147) identified were (i) unmodified MGMT-ASP (GNPVPILIPCHR; m/z 1315.73), (ii) methylated MGMT-ASP (GNPVPILIPMe-CHR; m/z 1329.74), and (iii) carboxymethylated MGMT-ASP (GNPVPILIPCM-CHR; m/z 1373.73).

Figure 3

MALDI-TOF mass spectra analysis of alkylated ASPs present after incubating his-MGMT with paired normal and tumor colorectal DNA. His-MGMT (50 pmol) was incubated with 2 mg of human colorectal DNA samples isolated from paired tumor (T) or normal (N) tissues numbered 19, 44, and 39 and unmodified CT-DNA. Peak intensities are shown in arbitrary units on the y-axis, and the intensity scale is the same for all panels. S/N > 10 for all detected alkylated MGMT ASPs. These included unidentified (U) ASPs with m/z values of 1459.7 (labeled U2) found in four samples, 1461.7 (labeled U3) in one sample, 1477.7 (labeled U4) in two samples, 1530.7 (labeled U5) in two samples, 1546.7 (labeled U6) in three samples, and 1555.7 (labeled U7) in four samples.