Abdelhady, R., Senthong, P., Eyers, C.E. et al. (11 more authors) (2023) 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. Chemical Research in Toxicology, 36 (12). pp. 1921-1929. ISSN 0893-228X
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2023 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Humans; Catalytic Domain; Colorectal Neoplasms; Cysteine; DNA; DNA Repair; Mass Spectrometry; O(6)-Methylguanine-DNA Methyltransferase; Oligodeoxyribonucleotides; Peptides |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > Department of Chemistry (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 05 Jan 2024 11:04 |
Last Modified: | 05 Jan 2024 11:04 |
Status: | Published |
Publisher: | American Chemical Society (ACS) |
Refereed: | Yes |
Identification Number: | 10.1021/acs.chemrestox.3c00207 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:207072 |