TY - JOUR
T1 - 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.
AU - Abdelhady, Rasha
AU - Senthong, Pattama
AU - Eyers, Claire E.
AU - Reamtong, Onrapak
AU - Cowley, Elizabeth
AU - Cannizzaro, Luca
AU - Stimpson, Joanna
AU - Cain, Kathleen
AU - Wilkinson, Oliver J.
AU - Williams, Nicholas H
AU - Barran, Perdita
AU - Margison, Geoffrey P.
AU - Williams, David
AU - Povey, Andrew
PY - 2023/12/18
Y1 - 2023/12/18
N2 - Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts were 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) and human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin and ASPs detected and quantified by matrix assisted laser desorption/ionization-time of flight mass spectrometry. ASPs containing S-methyl, -ethyl, -propyl, -hydroxyethyl, -carboxymethyl, -benzyl, and -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 characterised. 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 on cancer pathogenesis.
AB - Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts were 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) and human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin and ASPs detected and quantified by matrix assisted laser desorption/ionization-time of flight mass spectrometry. ASPs containing S-methyl, -ethyl, -propyl, -hydroxyethyl, -carboxymethyl, -benzyl, and -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 characterised. 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 on cancer pathogenesis.
KW - O6 -alkylguanine
KW - DNA adducts
KW - MGMT
KW - colorectal cancer
KW - mass spectrometry
UR - http://dx.doi.org/10.1021/acs.chemrestox.3c00207
U2 - 10.1021/acs.chemrestox.3c00207
DO - 10.1021/acs.chemrestox.3c00207
M3 - Article
SN - 0893-228X
VL - 36
SP - 1921
EP - 1929
JO - Chemical Research in Toxicology
JF - Chemical Research in Toxicology
IS - 12
ER -