Itraq reagent 4 plex kit

After digestion, peptides were vacuum centrifuged until dryness and redissolved in 0.5 M TEAB. iTRAQ labeling was performed using iTRAQ reagent 4-plex kit(AB Sciex Pte. Ltd) based on the manufacturer's protocol: allow iTRAQ® Reagents– 4 plex to reach room temperature, to each iTRAQ Reagent– 4 plex, add 50 μL of isopropanol; Vortex and then spin. transfer the contents of one iTRAQ Reagent– 4 plex vial to one sample tube; Vortex to mix, spin; If pH is <7.5, add up to 5 μL Dissolution Buffer, then incubate at room temperature for 2 hours, then the labeled peptides were obtained. Our samples were labeled as R2P2_1_113, R2P2_1_119, R2P2CMS_1_114 and R2P2CMS_2_121. Labeled peptides were then fractionated using strong cation-exchange (SCX), SCX chromatography was performed using a Shimadzu LC-20AB HPLC Pump system.

The iTRAQ and MS/MS analysis were performed in compound 2-treated MCF-7 cells. In simple terms, cells were dissolved in lysis buffer containing protease inhibitor (Sigma). Centrifuging the lysate for 1 h at 15 °C to get the supernatant, and stored it at −80 °C for further use. Protein quantitation was performed using Bio-Rad DC protein assay Kit (Bio-Rad). iTRAQ labeling was carried out using iTRAQ Reagent 4-Plex kit (AB SCIEX) based on the manufacturer’s protocol with minor modifications. The whole cell lysates were labeled with iTRAQ labeling reagent 114 and 115 for the pair of control MCF-7 and compound 2-treated MCF-7, respectively. After 2D LC analysis and tandem mass spectrometry analysis, protein identification and relative iTRAQ quantification were performed with ProteinPilot™ Software 4.2 (AB SCIEX) using the Paragon™ algorithm for the peptide identification, which was further processed by Pro GroupTM algorithm where isoform-specific quantification was adopted to trace the differences between expressions of various isoforms. Results with iTRAQ ratio cutoff values of 1.2 and 0.8 for fold-change and number cutoff values of 3 for quantifiable peptides for in protein abundance were accepted. Moreover, the results of 114 and 115 were adopted when their expression level were changed at the same trend.