Five micrograms of total tRNA was treated with Benzonase nuclease (Sigma), bacterial alkaline phosphatase (Invitrogen), and phosphodiesterase in the presence of deaminase inhibitors (0.5 μg/ml coformycin and 5 μg/ml tetrahydrouridine) and antioxidants (50 μM desferrioxamine and 50 μM butylatedhydroxytoluene) at 37°C overnight to generate ribonucleoside products. Proteins were removed from the digested product by filtration with 10K MWCO columns (Ambion). The ribonucleosides were fractionated by using a reversed-phase column (Thermo Hypersil Gold aQ column) with a gradient made of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) prior to introduction into an electrospray ionization triple quadrupole mass spectrometer (Agilent 6470), which was operated in the positive ion mode as described previously (27 (link),28 (link)). The modified ribonucleosides were identified by their HPLC retention times, CID fragmentation patterns, fragmentor voltages, and collision energies in comparison with available chemical synthetic standards. The level of each modified ribonucleoside was quantified from the MRM signal intensity and normalized by dividing by the summed signals of adenosine, guanosine, cytidine and uridine from the sample.