According to the instructions of the iTRAQ kit (Applied Biosystems, Carlsbad, CA, United States), 100 μg of the reduction reagent was added to each sample followed by incubation at 60°C for 1 h. Next, cysteine was added as a blocking reagent. After incubation for 10 min at room temperature, precooled acetone (acetone: sample = 5:1, v/v) was added to each tube followed by precipitation at −20°C for 1 h. Next, the samples were centrifuged at 12,000 rpm for 20 min at 4°C, and 20 μL of the dissolution solution was added to the precipitate to resuspend and dissolve the sample. Trypsin (Sigma, CA, United States) was added at a ratio of 1:20 (enzyme: protein), and this mixture was incubated overnight at 37°C. Labeling reagent was added to each tube and mixed. After reaction of the samples at room temperature for 1 h, 3 volumes of water were added.
The peptide mixture was then fractionated on a surveyor HPLC system with a 1.7 μm Waters BEH C18 2.1 × 50 mm reversed-phase column (Waters Company, CA, United States). The chromatographic method was as follows: the peptides were eluted with a 5–35% B (A: 20 mM ammonium formate, pH = 10.0; B: acetonitrile) gradient for 16 min. Absorbance was measured at 214 nm, and a total of 40 fractions were collected and then combined into 10 fractions.
The peptide mixture was then fractionated on a surveyor HPLC system with a 1.7 μm Waters BEH C18 2.1 × 50 mm reversed-phase column (Waters Company, CA, United States). The chromatographic method was as follows: the peptides were eluted with a 5–35% B (A: 20 mM ammonium formate, pH = 10.0; B: acetonitrile) gradient for 16 min. Absorbance was measured at 214 nm, and a total of 40 fractions were collected and then combined into 10 fractions.