Synergi fusion reversed phase column

Ribonucleosides were resolved with a Phenomenex Synergi Fusion reversed-phase column (100 × 2 mm, 2.5 μm particle size, 100 Å pore size) eluted with the following gradient of acetonitrile in 5 mM ammonium acetate (pH 5.3) at a flow rate of 0.35 ml/min and 35 °C: 0–1 min, 0%; 1–10 min, 0–10%, 10–14 min, 10–40%, 14–15 min, 40–80%. The HPLC column was coupled to an Agilent 6430 triple quadrupole mass spectrometer with an electrospray ionization source operated in positive ion mode with the following parameters: gas temperature, 350 °C; gas flow, 10 l/min; nebulizer, 45 psi; and capillary voltage, 3500 V. The first and third quadrupoles (Q1 and Q3) were fixed to unit resolution and the modifications were quantified by pre-determined molecular transitions. The dwell time for each ribonucleoside was 500 ms. The retention time, m/z of the transmitted parent ion, m/z of the monitored product ion, fragmentor voltage, and collision energy of each modified nucleoside are as follows: m¹A, 3.6 min, m/z 282→150, 100 V, 16 V; m¹G, 6.1 min, m/z 298→166, 90 V, 10 V; m¹I, 5.9 min, m/z 283→151, 80 V, 10 V; m²²G, 7.8 min, m/z 312→180, 100 V, 8 V. Modified ribonucleosides were identified using commercial standards. Three independent tRNA replicates were used to test AlkB demethylation efficiencies in this study.