TMT labelling and peptide fractionation

1.5 μg trypsinized peptides were dissolved in 50 mM TEAB pH 8.5 and labelled using the TMT10plex Isobaric Labeling Reagent Set (Thermo Fisher Scientific, 90111). Each sample was labeled with TMT10plex reagents at a 20:1 label:peptide w/w ratio, for 1 hour at room temperature. TMT labeling reaction was quenched with 0.2% hydroxylamine for 15 minutes at room temperature. The samples were pooled on ice, flash frozen and lyophilized. Pooled TMT-labelled peptides were cleaned and fractionated using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, 84868) according to manufacturer’s instruction for TMT experiments. After fractionation, samples were flash frozen and lyophilized.

For quantitative polysome profiling experiments, we performed 2 separate TMT10plex experiments. Each experiment contained interphase and mitotic, free, 40S, 60S, and 80S peptides. The two replicates were processed and analyzed independently.

For quantitative eIF1 interaction experiments, a single TMT experiment included 2 biological replicate GFP immunoprecipitations from GFP, GFP-eIF1, and NLS-GFP-eIF1 expressing cells.