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

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.

ESI MS spectra of modified 23-mer ODNs S3 Figure s2 MALDI-ToF mass spectral analysis of tryptic digests of MGMT incubated with different O 6 -alkylG containing ODNs.

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Figure s3 LC-Vion IMS QToF analysis of purified His-MGMT after in-solution tryptic digestion.

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Figure s4 LC-Vion IMS QToF analysis of His-MGMT tryptic digest following incubation with SS ODN containing O 6 -MeG.

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Figure s5 LC-Vion IMS QToF analysis of His-MGMT tryptic digest following incubation with control SS ODN (not containing O 6 -MeG).

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Figure s6 LC-Vion IMS QTof analysis of His-MGMT tryptic digest following incubation with control SS ODN spiked with synthetic methylated ASP.

Figure s7
Quantitation of MGMT ASPs.S9 Table s1 ODNs(G) +His-MGMT shows the identification of the carbamidomethylated form of ASP with a mass error of 3 ppm and 12 matched first generation primary ions S10 Table s2 ODNs(O 6 -MeG)+His-MGMT shows the identification of the methyl form of ASP with a mass error of 3 ppm and 15 matched first generation primary ions, the identification of the carbamidomethylated form of ASP has a mass error of 3 ppm and 8 matched first generation primary ions.

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Table s3 ODNs(G)+His-MGMT+Spiked MeASP indicate the methyl form of the spiked ASP with a mass error of 2.4 ppm and 17 matched first generation primary ions while the identification of the carbamidomethylated form of ASP has a mass error of 2.7 ppm and 7 matched first generation primary ions.Table s1 ODNs(G) +His-MGMT shows the identification of the carbamidomethylated form of ASP with a mass error of 3 ppm and 12 matched first generation primary ions.

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Table s2 ODNs(O 6 -MeG)+His-MGMT shows the identification of the methyl form of ASP with a mass error of 3 ppm and 15 matched first generation primary ions, the identification of the carbamidomethylated form of ASP has a mass error of 3 ppm and 8 matched first generation primary ions.

Figure
Figure s3 LC-Vion IMS QToF analysis of purified His-MGMT after in-solution tryptic digestion.A. HPLC elution profile of His-MGMT after tryptic digestion.BPI (base peak ion) chromatogram showing the elution profile with peaks of the digested peptides, including the one specific for MGMT-ASP at 41.77 min.Blue peaks are identified peptides while orange peaks are unidentified.Please see materials and methods section for HPLC details.The carbamidomethyl C modification is due to alkylation by iodacetamide during the insolution digestion procedure.B. Fragmentation pattern of MGMT ASP.Identification of MGMT-ASP with its fragmentation pattern, showing the intensities, Y axis, and the Mass (Da), X axis, of all the fragments obtained by the Vion IMS QToF from the ASP at 41.77 min, whose sequence is in Panel A.

Figure
Figure s5 LC-Vion IMS QToF analysis of His-MGMT tryptic digest following incubation with control SS ODN (not containing O 6 -MeG).SS ODN not containing O 6 -MeG (37.5 nmol) as negative control was incubated with His-MGMT (50 pmol).A: HPLC elution profile (zoom) of trypsin digested His-MGMT after incubation with SS ODNs not containing O 6 -MeG: BPI chromatogram showing the elution profile between 14-58 min with peaks of the digested peptides, indicating a specific peak of the carbamidomethylated MGMT-ASP (retention time 41.71 min), indicating, as expected, the absence of methylation as His-MGMT was incubated with control SS ODN lacking O 6 -MeG.Consequently, the ASP identified from the tryptic digestion of His-MGMT was all found as carbamidomethyl C. Blue peaks are identified peptides while orange peaks are unidentified.See materials and methods section for HPLC details.

Figure
Figure s6 LC-Vion IMS QTof analysis of His-MGMT tryptic digest following incubation with control SS ODN spiked with synthetic methylated ASP.Control SS ODN (37.5 nmol) was incubated with His-MGMT (50 pmol).Methylated synthetic ASP (112 nmol) was spiked in as a positive control.A: HPLC elution profile (zoom) of trypsin digested His-MGMT after incubation with SS ODNs not containing O 6 -MeG, spiked with synthesised methylated ASP.BPI chromatogram showing the elution profile between 14-58 min with peaks of the digested peptides, including specific peaks of both the spiked methylated synthetic MGMT-ASP (retention time 46.36 min), and of carbamidomethylated His-MGMT-ASP (retention time 41.75 min).Blue peaks are identified peptides while orange peaks are unidentified.See materials and methods section for HPLC details.B: Fragmentation pattern of MGMT spiked with synthetic Me-ASP.Identification of the spiked synthetic Me-ASP with its fragmentation pattern showing the intensities, Y axis, and the Mass (Da), X axis, of all the fragments obtained by the Vion IMS QTof.

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