Tq ms triple quadrupole lc spectrometer

RNA probes, total RNAs, and mRNAs were digested into nucleosides and the amount of a⁴C was measured by reverse-phase UHPLC on a T3 column with online MS detection using a Waters TQ MS triple-quadrupole LC spectrometer in positive electrospray ionization mode and was calculated based on the standard curve generated by pure standards. For each sample, around 500 ng RNA was digested by using 1 U nuclease P1 (Wako) in 30 μL reaction mixture containing 20 mM NH₄OAc at 42 °C for 2 h. Afterwards, 1 μL rSAP (NEB) and 3.5 μL Cutsmart buffer (NEB) were added and the reaction mixture was incubated at 37 °C for 2 h. Samples were filtered through a 0.22 μM filter (Millipore) and diluted to 100 μL. A 2 μL volume of the solution was injected into UHPLC-QQQ-MS/MS. The a⁴C nucleoside was quantified by using the nucleoside to base ion mass transition of 284 to 152.

All chemicals and reagents were used as purchased, without further purification. The cell lines utilized in this study (human HEK293T and HeLa cells) were obtained from the American Type Culture Collection (ATCC). ¹H and ¹³C NMR spectra were measured on a Bruker AVANCE III 400 NMR spectrometer using DMSO-d₆ as the solvent. High-resolution mass spectra (HRMS) were recorded on an AB Triple TOF 5600 plus (AB SCIEX, Framingham, USA) Mass spectrometer. HPLC profiles were acquired using a Waters e2695 module (flow rate of 3 ml min⁻¹) with an Atlantis T3 OBD Prep Column (100 Å, 5 μm, 10 mm × 250 mm, 1 per pkg; cat. no. 186008205). Human methyltransferase like 15 (METTL15),^{40 (link)} human methionine adenosyltransferase (MAT2A)^{47 (link)} and 5′-methylthioadenosine (MTN)^{37 (link)} were expressed and purified using the previously published procedures. Se-allyl-l-selenohomocysteine was synthesized following the previously reported protocol.^{12 (link)} UHPLC-QQQ-MS/MS results were acquired using a Waters TQ MS triple-quadrupole LC spectrometer. Ultracentrifugation was carried out using an Optima L-90K ultra centrifuge supplied with an SW40Ti swing rotor.