| Description |
Fragmentation processes of uridine-(TMS)(,4) were investigated using selectively deuterium and ('18)O labeled uridine. Decomposition pathways of uridine-(TMS)(,4) were measured by two metastable ion techniques, and plausible ion structures have been deduced from isotopic labeling data and mechanistic considerations. In addition, 128 silylated nucleoside mass spectra were examined to detect common characteristics for each class of nucleosides, for example, C-C versus C-N glycosidic bond, purine versus pyrimidine nucleosides, and (alpha) versus (beta) anomer. An Application of the knowledge of nucleoside fragmentation processes was shown by the structural characterization of a modified nucleoside isolated from Halococcus morrhuae tRNA. The utility of this knowledge for establishing the location of substituents and isotopic labels in the nucleoside skeleton was also described. The use of high resolution mass spectrometry to analyze and identify nucleoside components in tRNA hydrolysates was studied. The optimal experimental conditions: method of ionization, rate of heating the sample without defocusing the ion source, the resolution required to unequivocally determine the existence of a modified nucleoside in the hydrolysates, the sensitivity of this methodology, and the tradeoff between resolution and sensitivity were investigated and described. The procedures involved for the analysis of the high resolution mass spectrometric data were discussed in detail. Using E. coli tRNA('Phe) and tRNA('Glu) as models, modified nucleosides which occur only once in the tRNAs were identified with as little as 1 (mu)g of the tRNA hydrolysate. Resolution of 20,000 (10% valley definition) was found to be the best for the analysis of tRNA hydrolysate mixtures in terms of both resolution and sensitivity. The applications of this methodology were shown by the identification of N-{(9-(beta)-D-ribofuranosyl)-2-methylthiopurin-6yl-carbamoyl}threonine in tRNA(,1)('Lys) and tRNA(,3)('Lys) of B. subtilis. The procedure and application of this technique for the analysis of tRNAs of unknown components were also described with a tRNA('Leu) isolated from Morris hepatoma as an example. |
