DETECTION OF RIBOSE-METHYLATED NUCLEOTIDES IN PYRODICTIUM-OCCULTUM TRANSFER-RNA BY LIQUID-CHROMATOGRAPHY FRIT-FAST ATOM BOMBARDMENT MASS-SPECTROMETRY

Ribose-methylated dinucleotides of the type NmpN' derived from digesti on of tRNA with RNase T-2 were separated and characterized by directly combined liquid chromatography-mass spectrometry (LC-MS) with a conti nuous-flow frit-fast atom bombardment (frit-FAB) interface. Prediction of NmpN' peaks was readily made by comparison of the LC profile with that of comparative nuclease P-1 digest. The identity of the candidate peaks including NmpN' was further recognized by the mass spectra, in which NmpN' showed intense molecular-related ions, in addition to sequ ence-specific fragment ions, to verify the chemical structures in both positive- and negative-ion modes. The method was applied to screening NmpN' (and NmpN'mpN'') in tRNA from the extremely thermophilic archae on Pyrodictium occultum.