Initial characterisation of the alkyl DNA adductome in human colorectal DNA by mass spectroscopic analysis of MGMT tryptic peptides


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Rasha Abdelhady1,Geoffrey Margison1,Perdita Barran2,David Wiliams3,Andrew Povey1
1Centre for Occupational and Environmental Health, Division of Population Health, Health Services Research and Primary Care, School of Health Sciences, Faculty of Biology Medicine and Health, Ellen Wilkinson Building, University of Manchester, Oxford Road, Manchester M13 9PL,2The Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, Faculty of Science and Engineering, University of Manchester, Princess Street, Manchester M1 7DN,3Centre for Chemical Biology, Department of Chemistry, Faculty of Science, University of Sheffield, Western Bank, Sheffield S10 2TN

Abstract

Background

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.  

Method

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.

Results

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.

Conclusion

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.