Synergy fusion rp

Ribonucleosides were separated using a Synergy Fusion RP, 2.5 µm particle size, 100 Å pore size, 100 mm length, 2 mm inner diameter from Phenomenex (Torrance, CA), on an Agilent 1290 series HPLC system equipped with a diode array detector. Mobile phase A was 5 mM ammonium acetate adjusted to pH 5.3 with glacial acetic acid and mobile phase B was pure acetonitrile. Gradient elution started with 100% A for one minute, increase to 10% B after 10 minutes, 40% after 14 minutes and regeneration of starting conditions with 100% A for 3 additional minutes. The flow rate was 0.35 mL/minute and the column temperature 35°C. For mass spectrometric measurements an Agilent 6490 Triple Quadrupole mass spectrometer set to dynamic multiple reaction monitoring (MRM) mode was used. The MS was operated in positive ion mode with the following parameters: electro-spray ionization (ESI-MS, Agilent Jetstream), Fragmentor Voltage (set in tunefile to) 250 V, Cell Accelerator Voltage 2 V, N2-Gas temperature 150°C, N2-Gas flow 15 L/min, Nebulizer 30 psi, Sheath gas (N2) temperature 275 °C, Sheath gas flow 11 l/min, Capillary 2500 V and Nozzle Voltage 500 V. The mass transition for each modified nucleoside and its isotopomers are found in Table S2.

Ribonucleosides were separated using a Synergy Fusion RP, 2.5 μm particle size, 100 Å pore size, 100 mm length, 2 mm inner diameter from Phenomenex (Torrance, CA, USA), on an Agilent 1290 series HPLC system equipped with a diode array detector. Mobile phase A was 5 mM ammonium acetate adjusted to pH 5.3 with glacial acetic acid and mobile phase B was pure acetonitrile. Gradient elution started with 100% A for one minute, increase to 10% B after 5 min, 40% B after 7 min which was maintained for an additional minute. From minute 8 to 8.5 starting conditions are re-established and equilibrated for two additional minutes. The flow rate was 0.35 ml/min and the column temperature 35°C. The effluent from the column was directed through the DAD before entering the Agilent 6490 Triple Quadrupole mass spectrometer in dynamic multiple reaction monitoring (MRM) mode. The MS was operated in positive ion mode with the following parameters: electro-spray ionization (ESI-MS, Agilent Jetstream), Fragmentor Voltage (set in tunefile to) 250 V, Cell Accelerator Voltage 2 V, N₂-gas temperature 150°C, N₂-gas flow 15 l/min, Nebulizer 30 psi, sheath gas (N₂) temperature 275°C, sheath gas flow 11 l/min, capillary 2500 V and nozzle voltage 500 V. The mass transitions for each modified nucleoside are found in Supplementary Table S3.

Ribonucleosides were separated using a Synergy Fusion RP, 2.5 µm particle size, 100 Å pore size, 100 mm length, 2 mm inner diameter from Phenomenex, on an Agilent 1290 series HPLC system equipped with a diode array detector. Mobile phase A was 5 mM ammonium acetate and mobile phase B was pure acetonitrile. Gradient elution started with 100% A for 1 min, increased to 10% B after 10 min, 40% after 14 min, 80% after 20 min and regeneration of starting conditions with 100% A for 5 additional minutes. The flow rate was 0.35 mL/min and the column temperature 35°C. The effluent from the column was directed through the DAD before entering the Agilent 6430 triple quadrupole mass spectrometer in dynamic multiple reaction monitoring (MRM) mode. The MS was operated in positive ion mode with the following parameters: electro-spray ionization (ESI-MS), fragmentor voltage (average) 80 V, cell accelerator voltage 2 V, N₂-gas temperature 350°C, N₂-gas flow 10 L/min, nebulizer 40 psi, capillary 3500 V.
Modified nucleosides were identified based on having both the correct retention time and mass transition. Under our chromatography conditions, t6A eluted at 8.6 min while ct6A eluted at 6.8 min. Mass transitions were m/z 413 → 281 for t6A and m/z 395 → 263 for ct6A, both corresponding to the neutral loss of ribose. ¹⁵N-2-Deoxyadenosine was added to each sample as an internal standard.