Structural characterization of modified nucleosides in tRNA hydrolysates by frit-fast atom bombardment liquid...

Feasibility for the structural characterization of modified nucleosides in transfer RNA at low microgram levels has been investigated by using continuous-flow frit-fast atom bombardment liquid chromatography/mass spectrometry (frit-FAB LC/MS). Sample of tRNA(Phe) from brewer's yeast (Saccharomyces cerevisiae) was used as a main model, and enzymatically hydrolysed by nuclease P1 and alkaline phosphatase. The resulting nucleoside mixture was separated by using a microbore reversed-phase LC column (150 mm x 0.5 mm i.d.) with an aqueous ammonium acetate-methanol gradient, and the mass spectra were acquired on both positive and negative ionization modes. The modified nucleosides were characterized by comparison of the relative LC elution times with authentic nucleosides, and further confirmed by the structural information from the frit-FAB mass spectra where both molecular and base ions were in general observed as intense peaks in both ionization modes. Typically, 0.06-0.2 A260 units (3-10 micrograms) of isoaccepting tRNA was enough to obtain full-scan mass spectra of modified nucleosides, often occurring at a frequency of one per tRNA molecule using positive ion detection. The LC/MS system was used to screen modified nucleosides in tRNA of the extremely thermophilic microorganism Pyrodictium occultum.