Quantification of pseudouridine level by LC-MS

RNA (300 ng) was digested using 4U of P1 nuclease (Sigma N8630) in 25 mM NaCl 2.5 mM ZnCl₂ for 2 h at 37°C, followed by the addition of 5 units of Antarctic Phosphatase (New England Biolabs). The mixture was incubated at 37°C for another 2 h. The reaction was analyzed using LC–MS/MS. The nucleosides were separated by ultra-performance liquid chromatography (Vanquish UHPLC Thermo Fisher scientific) on a Hyprsil GOLD aQ column (Thermo Scientific 25303-152130), and then detected by Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific) in Parallel Reaction Monitoring Mode (PRM). The mass transitions of m/z 243.06 to 153.03, 183.04 (pseudouridine), m/z 243.06 to 110.02, 82.02 (U) in the negative mode, and mass transitions of m/z 268.10 to 136.06, (A), m/z 284.1 to 152.06 (G) and m/z 244.1 to 112.05 (C) in the positive mode were monitored and recorded. Concentrations of nucleosides in RNA samples were deduced by fitting the signal intensities using standard curves.