Measurement of O6-alkylguanine-DNA alkyltransferase activity in tumour cells using stable isotope dilution HPLC-ESIMS/MS

摘要: Abstract The repair of DNA mediated by O6-alkylguanine-DNA alkyltransferase (AGT) provides protection against damage from endogenous or exogenous alkylation the O6 position guanine. However, this acts as a double-edged sword in cancer treatment, it not only protects normal cells chemotherapy-associated toxicities, but also results cell resistance to guanine O6-alkylating antitumour agents. Thus, AGT plays an important role predicting individual susceptibility carcinogens and chemotherapies. Accordingly, is necessary establish quantitative method for determining activity with high accuracy, sensitivity practicality. Here, we describe novel nonradioactive measuring using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI⿿MS/MS). This based on irreversibility removal O6-alkyl group affinity O6-benzylguanine (O6-BG) substrate. HPLC-ESI⿿MS/MS was used measure activities protein extracts eight tumour lines, demonstrating that quite variable among different ranging nondetectable 1021 fmol/mg protein. experiments performed intact yielded similar exhibited slightly higher than those observed extracts. accuracy confirmed examination expression levels western blotting analysis. To our knowledge, first spectrometry-based assay, will likely provide assistance screening risk application