proteome analysis using mass spectrometry (MS) in acute shYthdf2-mediated knockdown of cultured RGCs

To identify mouse retinal proteomics changes after Ythdf2 knockdown, we extracted the proteomics of E15.5 retina neurons which were cultured and infected with lentiviral shYthdf2 or shCtrl by urea. After using 100 mM TEAB (Sigma) to dilute the urea concentration to less than 2 M in each sample, trypsin (Promega) was then added to digest the proteins overnight at 37 °C. Peptides were further desalted by Strata X C18 SPE column (Phenomenex) and labelled with TMT10plex Mass Tag Labelling kit (Thermo Scientific) according to the manufactural instructions. Finally, the labeled peptides were subjected to HPLC fractionation and LC-MS/MS analysis.