Protein extraction, reduction, alkylation, digestion, TMT labeling and LC–MS/MS

Protein extraction was carried out according to a previously described method⁵⁴, and the final protein concentration was quantified using the BCA (Beyotime Biotechnology, Beijing, China) assay as per the manufacturer’s guidelines. The proteins’ disulfide bonds of the samples were broken by mixing with a solution of 10 mM DTT for 1 h. Thereafter, these samples were incubated in 50 mM iodoacetamide for alkylation at room temperature for 1 h away from light. Trypsin was used for protein digestion at a volume ratio of 1:50 at 37 °C for 14 h and 1 μL of formic acid was added to the solution for stopping the enzymatic digestion. Finally, peptides were concentrated using a SpeedVac system (Marin Christ, Osterod, Germany). The sample peptide was labeled with TMT kits (Thermo Fisher Scientific, USA) as per the manufacturer’s guidelines. The samples were then combined and stored at -80 °C until the following LC/MS analysis. the peptide sample was loaded onto an LC–MS system. The peptide enrichment, eluted, MS/MS data collected and saved according to a previously study⁵⁴. Data are available via ProteomeXchange with identifier accession PXD022768.