The isolated tRNAs were digested with RNase T1 (Ambion) and analyzed by capillary liquid chromatography (LC)/nano-electron spray ionization mass spectrometry (ESI-MS), as described (9 (link),42 (link),43 (link)). SI-labeled nucleoside analysis by LC/MS was performed as described (44 (link),45 (link)). For nucleoside analysis of HeLa cells cultured under taurine-depleted condition, 5 μg of tRNA fraction was digested as follows: 10 μl reaction mixture consisting of 20 mM ammonium acetate (pH 5.3) and 0.01 units/μl nuclease P1 (Wako Pure Chemical Industries) was incubated at 37°C for 30 min, followed by adding 0.5 μl of 1 M ammonium bicarbonate (pH 8.0) and 0.25 units of phosphodiesterase I (Worthington Biochemical Corporation) and incubated at 37°C for 30 min, then 0.1 units of alkaline Phosphatase (Escherichia coli C75) (Takara Bio) was added and further incubated at 37°C for 30 min. The digests were subjected to hydrophilic interaction liquid chromatography (HILIC)/MS analysis as described (46 (link)). For detection of THF derivatives bound to Thermotoga maritima MnmE, 175 μg of recombinant protein was directly injected into the LC/MS under the same conditions used for the nucleoside analysis.