ABSTRACT
B. Long Synthetic Oligonucleotides (More Than 50 Bases) A first class of long oligonucleotides which have been analyzed by ESI-MS are transfer RNAs (tRNAs) [14]. These tRNAs had lengths of 74-88 bases and were isolated from ribosomal subunits from E. coli cells. In order to determine these tRNAs with ESI-MS, the cation adducts had to be removed. The analyte ion current from the oligonucleotide increased 10-fold after three ethanol precipitations from 2.5M ammonium acetate compared to the signal obtained after only one precipitation. However, the peak widths did not decrease with the increasing number of ammonium acetate precipitations [14], proving precipitation alone to be insufficient to remove cation-adduct species and to provide accurate mass values. Sample losses could be minimized, and sensitivity and accuracy were greatly enhanced by addition of the chelating agent CDTA (2.5 nmol to 300 pmol tRNA) and TEA (10 )lL 0.1% to 300 pmol tRNA) after only one ammonium acetate precipitation. The chelating agent removes divalent cation adducts such as Mg2+, and TEA removes monovalent cation adducts such as N a+ and K+. As can be seen from Fig. 6, the addition of both CDT A and TEA is required for optimal results, and not only is the signal-to-noise ratio enhanced but also the peak widths are dramatically decreased, leading to a better mass accuracy of less than 100 ppm. The determination of the molecular mass of these tRNAs not only allowed the determination of their length but also made it possible to identifY the tRNAs and gave an indication of their base composition.
