Abstract 4242: Mass spectroscopic analysis of MGMT tryptic peptides allows detection of O6-alkylguanine adducts in oligodeoxyribonucleotides, temozolomide modified calf thymus DNA and human colorectal cancer DNA

Abstract

Abstract Background: Red and processed meat consumption increases human colorectal cancer (CRC) risk, potentially by heme-catalysed formation of carcinogenic N-nitrosocompounds (NNOC) that form mutagenic O6-alkylguanine (O6-alkG) DNA adducts. NNOC formation cannot be accurately estimated from meat consumption alone due to in situ processing required for their formation. We are thus developing a novel approach to assess NNOC exposure by quantifying O6-alkylguanine DNA adducts that result from their exposure, using the known action of the DNA repair protein, O6-alkylguanine O6-alkyltransferase (MGMT), to irreversibly transfer the O6-alkyl to an active site cysteine residue in MGMT. Methods: Oligodeoxyribonucleotides (ODNs) containing O6-methylguanine (O6-MeG), O6-carboxymethylguanine (O6-CMG), O6-carboxyethylguanine (O6-CEG) were synthesised by alcohol modification of an ODN containing the convertible base 2-amino-6-methylsulfonylpurine. O6-MeG containing calf thymus (CT) DNA was prepared by incubation with temozolomide (TMZ). Both maltose binding protein-MGMT (MBP-MGMT) and his-tagged MGMT (his-MGMT) fusion proteins were expressed, purified, and MGMT functional activity determined by tritium transfer using CT DNA methylated with [3H]-N-Nitrosomethylurea. Double stranded O6-alkG-containing ODNs, unmodified ODN and methylated CT DNA were incubated with MBP-MGMT and the resulting alkylated MGMT digested with trypsin and analysed by mass spectrometry using MALDI-TOF. Human CRC DNA samples were incubated with his-MGMT which was then recovered using Ni-coated magnetic beads. MGMT was digested in situ with trypsin and the tryptic peptides analysed by MALDI-TOF. Results: S-methylcysteine (m/z 1329.7), S-carboxymethylcysteine (m/z 1373.7) and S-carboxyethylcysteine, modified MGMT active site peptides were detected in tryptic digests of MBP-MGMT incubated with O6-MeG-, O6-CMG- and O6-CEG-containing ODNs respectively. Only the unmodified active site peptide (m/z 1315.7) was found after incubating MGMT with unmodified ODN. Subsequent MS analysis of tryptic digests of MGMT incubated with TMZ modified CT DNA also revealed the active site peptide containing S-methylcysteine. Using this approach, pilot studies revealed the presence of O6-MeG, O6-CMG and O6-CEG in CRC DNA. Conclusions: These results demonstrate proof of principle and confirm that this approach could be used to characterise O6-alkylguanine adducts in human CRC DNA. Citation Format: Rasha A. Abdelhady, Perdita E. Barran, David M. Williams, Andrew C. Povey. Mass spectroscopic analysis of MGMT tryptic peptides allows detection of O6-alkylguanine adducts in oligodeoxyribonucleotides, temozolomide modified calf thymus DNA and human colorectal cancer DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4242. doi:10.1158/1538-7445.AM2017-4242