Detection of ribose-methylated nucleotides in Pyrodictium occultum tRNA by liquid chromatography--frit-fast atom bombardment mass spectrometry.

Ribose-methylated dinucleotides of the type NmpN' derived from digestion of tRNA with RNase T2 were separated and characterized by directly combined liquid chromatography--mass spectrometry (LC-MS) with a continuous-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 P1 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 sequence-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 archaeon Pyrodictium occultum.