RNA Nucleoside Mass Spectrometry Protocol

RNA nucleoside mass spectrometry was performed as previously described in refs. ^{45 (link)–47 (link)}. A 25 μL solution containing 3 μg of heart total RNA, 20 mM Hepes-KOH (pH 7.6), 2 units of Nuclease P1 (Fujifilm, Tokyo, Japan), and 0.25 units of bacterial alkaline phosphatase (Takara, Kusatsu, Japan) was incubated at 37 °C for 3 h. About 3 μL of the nucleoside solution was then injected into the LC-MS-8050 system (Shimadzu, Kyoto, Japan). The nucleosides were first separated by an Inertsil ODS-3 column (GL Science, Tokyo, Japan) using a mobile phase that continuously changed from 100% of solution A (5 mM ammonium acetate in water, pH 5.3) to 100 % of solution B (60% acetonitrile in water) in 17 min at a flow rate of 0.4 mL min⁻¹, followed by electrospray ionization and a triple quadrupole mass spectrometry in the multiple reaction monitoring modes.