2.3. Protein Extraction and Isobaric Labeling for Proteomics Analysis

MCF-7 treated with and without hesperidin and chlorogenic acid were collected and homogenized in lysis buffer (8 M urea in 50 mM triethyl ammonium bicarbonate (TEAB) buffer, pH 8) to extract proteins. Cell lysate was centrifuged at 16,000× g for 10 min at 4 °C, the supernatants were collected, and the protein concentration was determined by Bradford assay (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. For reduction and alkylation of disulfide bonds in proteins, 100 μg of protein was treated with dithiothreitol (DTT) to a final concentration of 10 mM and then incubated at 55 °C for 30 min. Then, iodoacetamide (IAA) was added to a final concentration of 20 mM prior to incubation for 30 min at room temperature in the dark. Then, a second aliquot of DTT was added to quench unreacted IAA. Six volumes of pre-chilled (−20 °C) acetone was added to each protein sample and frozen at −20 °C for at least 4 h to precipitate proteins. The acetone-precipitated protein pellet was collected by centrifugation, resuspended with 50 mM triethylamonium bicarbonate (TEAB), and followed by the addition of trypsin (protein/trypsin ratio of 50:1) for digestion overnight at 37 °C. The peptide samples were labeled using a Tandem Mass Tag (TMT) six-plex isobaric label reagent set (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. Each peptide solution was incubated for 1 h at room temperature and quenched for 15 min with 8 μL of 5% hydroxylamine solution. After labeling, the samples were desalted by Oasis HLB solid-phase extraction cartridges (Waters Corporation, Milford, MA, USA). Briefly, the cartridges were wet by acetonitrile followed by washing with 0.1% formic acid solution. The TMT-labeled peptide samples were loaded onto cartridges. After washing by 0.1% formic acid solution twice, peptides were eluted by 5% formic acid in 50% acetonitrile three times and dried by SpeedVac vacuum concentrators (SPD110, Thermo Fisher Scientific Inc.). In order to reduce the complexity and improve the protein identification and confidence level of quantification, the SpeedVac-drived TMT-labeled peptides were divided into 8 fractions by using a high-pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific Inc.). Fractions were dried by vacuum concentrators and dissolved in 0.1% formic acid before Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) analysis.