Analysis of mRNA Modifications via LC-MS/MS

Total RNA was extracted from each sample using TRIzol reagent/RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA was quantified and qualified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States), NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Waltham, MA, United States), and 1% agarose gel. mRNA was isolated from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490; NEB, Ipswich, MA, United States) according to the manufacturer’s protocol. Purified mRNA was hydrolyzed to single nucleosides which were then dephosphorylated by enzyme treatment. Pretreated nucleosides solution was deproteinized using a Sartorius 10,000 Da molecular weight cut-off spin filter (Göttingen, Germany). Analysis of nucleoside mixtures was performed using an Agilent 6460 QQQ MS with ESI Jetstream ionization operated in positive ion mode, with SB-Aq 3.5 μm 2.1 × 150 mm high-performance LC column (Agilent Technologies) conditions according to the following solvent gradient [Solution A, high-performance LC-grade water with the relevant amount of formic acid to obtain a final formic acid concentration of 0.1% (vol/vol); Solution B, 100% acetonitrile with the relevant amount of formic acid to achieve a final formic acid concentration of 0.1% (vol/vol)]. The voltages and source gas parameters were as follows: gas temperature, 350°C; gas flow, 7 l min^–1; nebulizer, 40 psi; sheath gas temperature, 350°C; sheath gas flow, 11 l min^–1; capillary voltage, 3,500 V; and VCharging, 500 V. The molecular transition ions were quantified in multiple reaction monitoring mode (Supplementary Table S2). Multiple reaction monitoring peak information of modified nucleosides for each sample was extracted using Agilent Qualitative Analysis software. Peaks with signal-to-noise ratio ≥ 5 were considered as detectable nucleosides. Peak areas were then normalized to the quantity of purified mRNA of each sample.