4.5. iTRAQ Labeling

After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for iTRAQ kit. Briefly, one unit of 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. Then, pooled proteins were then labeled with iTRAQ reagents as follows M12CK: 113 and 114 (control), M12W: 115 and 116, M25CK: 117 and 118, and M25W: 119 and 121, and incubated at room temperature for 2 h. All labeled peptides were then combined before being dried in a vacuum concentrator (Eppendorf Concentrator 5301, Hamburg, Germany).