2.4.2. TMT Labeling and LC-MS/MS Analysis

The peptides were desalinated and dried, and each sample was added with 200 mM TEAB for dissolution. Then, the peptides were labeled according to the instructions of the TMT kit. After being incubated at room temperature for 1 h, the TMT-labeled peptides were mixed, desalted, and dried and redissolved in 100 μL 0.1% FA. High-performance liquid chromatography (HPLC) was used to separate TMT-labeled peptides based on specific components. Briefly, labeled peptides were loaded onto the Xbridge BEH300 C18 column (Waters, USA) for separation of peptide samples with UltiMate 3000 UHPLC (Thermo Fisher Scientific, USA) and separated into 15 fractions. Lastly, the fractions were dried and then dissolved in 20 μL 0.1% FA followed by liquid chromatography- (LC-) mass spectrometry (MS)/MS analysis. The precursor ion mass tolerance was set to 20 ppm for all mass spectra obtained in the Orbitrap mass analyzer, and the fragment ion mass tolerance was corrected to 20 MMU for all MS2 spectra obtained. The row data were searched against the database of UniProt mouse FASTA with Proteome Discoverer software. The up- and downregulation of protein expression was set at ratio ≥ 1.2 and ≤0.83. All the proteomic data were deposited with the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022422.