Cells were cultured for 48 h, the culture medium was aspirated, and the cells were rinsed 3 times with ice-cold phosphate buffer solution (PBS). The cells were harvested and softly homogenized in an ice-cold RIPA Lysis Reagent (BOSTER, Wuhan, China) containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktail (BOSTER, Wuhan, China), sonicated for 400 W × 4 min (6 s on, 4 s off), and centrifuged at 20,000 × g for 15 min. The supernatant containing the total MCF-7 cell proteins was precipitated with 5 volumes of ice-cold ethanol/ acetone/acetic acid (50/50/0.1, v/v/v) at −20 °C for 2 h. Protein precipitant was centrifuged at 20,000 × g for 1 h. The pellet was washed separately with 1 ml of acetone and 75% ethanol, and redissolved in 600 μL of 6 M guanidine hydrochloride, 100 mM ammonium bicarbonate solution, the protein concentration was determined by Bradford Protein Assay Kit (BOSTER, Wuhan, China) according to the manufacturer’s instructions. The protein concentration of each sample was diluted to 3.00 mg/mL with 6 M guanidine hydrochloride, 100 mM ammonium bicarbonate solution. Cysteine bonds were reduced with 20 mM DTT (dithiothreitol) (Sigma-Aldrich) for 1 h at 56 °C and alkylated with 60 mM IAA (iodoacetamide) (Sigma-Aldrich) for 40 min at room temperature in darkness, and the resulting mixed solution was transferred into ultrafiltration units (10 K, PN: VN01H02, Sartorious). After 30 min of centrifugation at 10,000 × g, the protein adhered to the filter membrane was rinsed 3 times with 0.5 M urea, 100 mM ammonium bicarbonate solution and incubated overnight with trypsin (enzyme-to-protein ratio = 1:50, w/w) in 0.5 M urea, 100 mM ammonium bicarbonate solution at 37 °C. The peptide samples in ultrafiltration units were collected by centrifugation at 10,000 × g for 30 min, and acidified to 1% formic acid. The resulting samples were transferred to 1.5 mL EP tubes for vacuum-dry at 25 °C, the residues were redissolved in loading buffer (65% acetonitrile/2% trifluoroacetic acid/ saturated by glutamic acid) for further TiO2 beads enrichment.