iTRAQ labeling and fractionation

iTRAQ labeling was performed according to the manufacturer’s instructions (Applied Biosystems, United States). Briefly, the peptide mixtures were reconstituted with 30 μl of iTRAQ dissolution buffer. The label method was performed for each sample (100 μg) using the iTRAQ Reagent-8plex Multiplex Kit (AB SCIEX, United States). The labeling solution reaction was then incubated at room temperature for 1 h prior to further analysis. The six labeled samples were pooled into one sample and dried in a vacuum centrifuge at room temperature. The iTRAQ-labeled peptides were subjected to High-pH Reversed-Phase Fractionation in a 1100 Series HPLC Value System (Agilent, United States) equipped with a Gemini-NX (Phenomenex, 00F-4453-E0) column (4.6 mm × 150 mm, 3 μm, 110 Å). The peptides were eluted at a flow rate of 0.8 ml/min. Buffer A consisted of 10 mm ammonium acetate (pH 10.0), and buffer B consisted of 10 mm ammonium acetate and 90% v/v CAN (pH 10.0). The following gradient was applied to perform separation: 100% buffer A for 40 min, 0–5% buffer B for 3 min, 5–35% buffer B for 30 min, 35–70% buffer B for 10 min, 70–75% buffer B for 10 min, 75–100% buffer B for 7 min, 100% buffer B for 15 min, and 100% buffer A for 15 min. The elution process was monitored by measuring and was finally combined into 15 pools. Each fraction was concentrated via vacuum centrifugation and reconstituted in 40 μl of 0.1% v/v trifluoroacetic acid. All samples were stored at −80°C until LC-MS/MS analysis.