2.5. Tandem Mass Tag (TMT) Labeling and HPLC Fractionation

After digestion, the samples were redissolved in 50 μL TEAB (100 mM). TMT labeling 6-plex kits (catalog no. 90064CH, Thermo Fisher Scientific, Waltham, MA, USA) were separately dissolved in 88 μL HPLC-grade acetonitrile (Thermo Fisher Scientific, Waltham, MA, USA). Each sample with 50 μL TEAB buffer was mixed with 41 μL of prepared TMT reagent at room temperature for 1 h. The reaction was then stopped by 8 μL 5% hydroxylamine (Sigma-Aldrich, Saint Louis, MO, USA). Finally, TMT-labeled peptides were dried by vacuum centrifugation.

TMA-labeled peptide mixtures were fractionated according to high pH reverse-phase HPLC method with an Agilent Zorbax Extend C18 column (5 μm, 150 mm × 2.1 mm). Mobile phase was made up of water with 2% acetonitrile (A) and 90% acetonitrile in water (B). Procedures of solvent gradient elution were as follows: 98% A for 0–8 min, 98–95% A for 8–8.01 min, 95–75% A for 8.01–48 min, 75–60% A for 48–60 min, 60–10% A for 60–60.01 min, 10% A for 60.01–70 min, 10–98% A for 70–70.01 min, and 98% A for 70.01–75 min. The peptides were separated at a delivery flow rate of 0.3 mL/min and detection wavelength was set, respectively, at 210 nm and 280 nm. Samples were collected from 8 to 60 min and the elution buffers were harvested into the centrifuge tube numbered from 1 to 15 at interval of 1 min. After collection, the separated peptides were freeze-dried in vacuum for further analysis.