Quantitative Analysis of tRNA Nucleosides

Total tRNA (300 ng) in aqueous digestion mix (30 μL) was digested to single nucleosides by using 2 U alkaline phosphatase, 0.2 U phosphodiesterase I (VWR, Radnor, Pennsylvania, USA), and 2 U benzonase in Tris (pH 8, 5 mM) and MgCl₂ (1 mM) containing buffer. Furthermore, 0.5 µg tetrahydrouridine (Merck, Darmstadt, Germany), 1 µM butylated hydroxytoluene, and 0.1 µg pentostatin were added to avoid deamination and oxidation of the nucleosides. When quantification of dihydrouridine was intended tetrahydrouridine was omitted. After incubation for 2 h at 37 °C, 20 µL of LC-MS buffer A (QQQ) was added to the mixture and then filtered through 96-well filter plates (AcroPrep Advance 350 10 K Omega, PALL Corporation, New York, USA) at 3000 × g and 4 °C for 30 min. A stable isotope labeled internal standard (SILIS) was produced in S. cerevisiae using ¹³C and ¹⁵N rich growth medium (Silantes, Munich, Germany, Product# 111601402) following recently described procedures^{16 ,31 (link)}. 1/10 Vol. of SILIS was added to each filtrate before analysis by QQQ mass spectrometry. For each sample 10 µL were injected (~60 ng of sample tRNA).