TMT Labeling and HPLC Fractionation

After trypsin digestion, the resulting peptide mixture was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide mixtures were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit. Briefly, one unit of TMT/iTRAQ reagent were thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted and dried by vacuum centrifugation.

The samples were fractionated according to the protocol (Pan et al., 2018). The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into nine fractions for proteomic analysis and six for phosphoproteomic analysis, and then dried by vacuum centrifuging.