Structural characterization of modified nucleosides in tRNA hydrolysates by frit-fast atom bombardment liquid chromatography/mass spectrometry

✍ Scribed by Naohito Takeda; Masashi Nakamura; Hideo Yoshizumi; Akira Tatematsu

• Publisher: John Wiley and Sons
• Year: 1994
• Tongue: English
• Weight: 864 KB
• Volume: 23
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/bms.1200230803

✦ Synopsis

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 (fnt-FAB LC/MS). Sample of tRNApbC from brewer's yeast (Sucdaromyces cerevisiae) was used as a main model, and enzymatically hydrolysed by nuclease P, and alkaline phosphatase. The resulting nucleoside mixture was separated by using a microbore reversed-phase LC column (150 mm x 0.5 mm id.) with an aqueous ammonium acetatemethanol 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 nuclee sides, 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. Tyically, 0.06-0.2 A,,, units (3-10 pg) 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 occulrum.