Proteomic Analysis of Ψ-Containing mRNAs

The Ψ-PTC mRNAs were compared to uridine containing (non-Ψ) PTC mRNAs (Supplementary Table S8). Flag-eGFP peptides translated in HEK-293T cells were purified with anti-Flag M2 magnetic beads (Sigma). Pulled down proteins were extensively washed with 50 mM ammonium acetate and were directly digested on the beads adding Trypsin and alkylated with iodoacetamide (55 mM). Peptides were analyzed using a Dionex, UltiMate 3000 nano-HPLC system (Germering, Germany) coupled via nanospray ionization source to a Thermo Scientific Q Exactive HF mass spectrometer (Vienna, Austria) using instrument settings as described previously (44 (link)). The database search was performed using ProteomeDiscover (Version 2.1, Thermo Scientific). Sensitivity of the assay was calculated by the median abundance of the 10 least abundant peptides in the least sensitive sample, out of the total abundance of all of the C-terminal peptides in any of the samples.