STUDIES OF METHYLATED NUCLEOSIDES BY TANDEM MASS SPECTROMETRY (GAS PHASE REACTIONS)

Currently, a great deal of effort is being devoted to the devel- opment of more selective and sensitive methods of analysis for carcinogen-methylated nucleosides in DNA. A major goal in studies of methylated nucleosides is to correlate the products and extent of methylation with cancer and tumor induction processes. In light of this, a tandem mass spectrometry (MS/MS) methodology has been developed which allows detection of methyldeoxyribonucleosides at high femtomole levels. Various ionization capabilities were evaluated for these studies; desorption chemical ionization was found to be the method of choice. MS/MS was used to detect naturally-occurring deoxyribonucleosides and methyldeoxyribonucleosides from intact DNA. In addition MS/MS was used to detect already isolated methyl- deoxyribonucleosides which occur naturally in DNA or which are produced by in vitro action of a carcinogen-methylating agent. Other applications of MS/MS include the differentiation of isomeric base-methylated methyldeoxyribonucleosides, using the daughter spectra of the corresponding protonated bases, and the characterization and detection of a phosphate-alkylated dinucleotide down to sub- picomole levels. The thesis also discusses gas phase methylation of dihydroxy- benzenes under chemical ionization conditions. Methyl fluoride and methyl chloride reagent gases are used to produce dimethylhalonium ions which react as methylating species. The reactions were carried in situ in a MS/MS instrument, and the products of methylation were characterized through comparisons of daughter spectra of the meth- ylated product ions with those of protonated standards (i.e. methoxy- phenols and toluene-dihydroxybenzenes). These studies and the proven capability of MS/MS in distinguishing isomeric methylde- oxyribonucleosides formed the basis for an investigation of the gas phase methylation of nucleosides. In these experiments, methylating ions derived from methyl chloride and methyl iodide attack the base moiety of deoxyguanosine, deoxycytidine, thymidine and deoxyade- nosine preferentially at positions N-7, N-3, O-4 and N-3, respectively. Notably, these results were found to correlate with those obtained from the in vivo and in vitro action of carcinogenic methylating agents on DNA