Protein Preparation and iTRAQ Labeling

The iTRAQ reagent multiplex kit was bought commercially (Applied Biosystems, Foster City, CA, United States). The cells of control and dioscin (5.0 μg/mL) groups with two cell culture replicates were harvested and washed with PBS. Then, the cells were treated with radioimmunoprecipitation assay lysis buffer (50 mM Tris–HCl, 1% SDC, 150 mM NaCl, 0.1% Triton X-100, pH 8.0) supplemented with 1 mM PMSF (phenylmethane sulfonyl fluoride). Next, protein solubilization was achieved by ultrasonic, and cellular debris was removed by centrifugation (12,000 × g, 20 min, 4°C). Then, protein concentration of the middle layer was quantified by BCA Protein Assay Kit. Further, two independent biological replicates (200 μg) of each group were prepared by FASP (filter aided sample preparation), and iTRAQ labeling was performed according to the manufacturer’s introduction (Applied Biosystems, Foster City, CA, United States). The protein samples of dioscin-treated group (5.0 μg/mL) were labeled with iTRAQ 117 and iTRAQ 118, and the protein samples of control group were labeled with iTRAQ 119 and iTRAQ 121. After that, the labeled digests were pooled and dried by vacuum centrifuge.