Structural studies of tRNAs by capillary high performance liquid chromatography/electrospray mass spectrometry

Capillary high performance liquid chromatography/ electrospray mass spectrometry (LC/ ESI/MS) was applied to studies of nucleic acids. Since the presence of salt adducts obscures the molecular peaks and hinders the precise determination of molecular mass, conditions were sought in which effective online desalting and efficient ionization could be achieved simultaneously. In the presence of triethylamine, which is routinely used for the reversed-phase separation of nucleic acid, small oligo-nucleotides could be measured satisfactorily, while RNA tended to show more pronounced adduct peaks. When tributylamine was used instead of the shorter analogue, the adduct peak formation was well suppressed, and the hydrophobic nature of the counter ion made the nucleotides binding to the column tighter, resulting in the better chromatographic separation. The efficient ionization and suppression of the ion adducts make it possible to determine precisely the molecular mass of large nucleotides such as tRNA.