Initial characterisation of the alkyl DNA adductome in human colorectal DNA by mass spectroscopic analysis of MGMT tryptic peptides
Session type: Proffered paper sessions
Red and processed meat consumption increases human colorectal cancer (CRC) risk, possibly by heme-catalysed formation of carcinogenic N-nitroso compounds (NNOC), from a diverse assortment of amines and related compounds. These NNOC can potentially generate a variety of alkyl DNA adducts including pro-mutagenic O6-alkylguanines (O6-alkylGs). This alkyl adductome has not yet been characterised and to investigate it, we have exploited the action of the DNA repair protein, O6-alkylguanine DNA-alkyltransferase (MGMT), which covalently transfers the alkyl group from O6-alkylGs to a cysteine residue at the active site. Following trypsin treatment, the alkyl modified active site peptide (ASP) can be detected by MALDI-ToF.
Chemically synthesised oligodeoxyribonucleotides (ODNs) containing O6-methylguanine (O6-MeG), O6-carboxymethylguanine (O6-CMG), O6-carboxyethylguanine (O6-CEG), or O6-hydroxyethylguanine (O6-HOEtG), Temozolomide modified Calf thymus DNA , and human CRC DNA samples were incubated with MGMT, digested with trypsin either directly (ODNs) or following purification using nickel-coated beads (DNA samples) and analysed by MALDI-ToF.
S-methylcysteine (m/z 1329.7), S-carboxymethylcysteine (m/z 1373.7), S-carboxyethylcysteine (m/z 1387.7) and S-hydroxyethylcysteine (m/z 1359.7) modified ASPs were detected in tryptic digests of MGMT after incubation with the corresponding ODN. Only unmodified MGMT-ASP (m/z 1315.7) was detected following MGMT incubation with unmodified ODN. Tryptic digests of MGMT incubated with (i) methylated calf thymus DNA contained methylated MGMT-ASP and (ii) human CRC DNA contained a range of alkyl modified ASPs including: S-methylcysteine (13/14 samples analysed), S-carboxymethylcysteine (8/14) and S-hydroxyethylcysteine (1/14). A further seven putative alkylated MGMT ASPs were detected: of which 2 were found in 4 DNA samples, 1 in 3 , 1 in 2 and 3 in just one DNA sample.
These results show that this novel approach can detect both known and as yet unidentified O6-alkylGs and confirm that human CRC DNA contains a range of O6-alkylG adducts that probably constitute only a fraction of the DNA alkyl adductome.