The iTRAQ-labeled peptides were fractionated by SCX chromatography using the AKTA Purifier system (GE Healthcare). In brief, the peptide mixture was reconstituted with 2 mL buffer A (10 mM KH2P04 in 25% of acetonitrile, pH 3.0), loaded into a polysulphoethyl 4.6 mm × 100 mm column (5 μm, 200 Å, PolyLC Inc, Maryland, USA.) and then eluted at a flow rate of 1 mL/min with a gradient of 0%–10% buffer B (10 mM KH2P04 pH 3.0, 500 mM KCl and 25% acetonitrile) for 2 min, 10% to 20% buffer B for 25 min followed by 20% to 45% buffer B for 5 min and 50% to 100% buffer B for 5 min. The UV absorbance at 214 nm was monitored when the fractions were collected. The fractions were collected every minute (about 30 fractions), combined in 10 pools and then desalted on C18 Cartridges [Empore SPE Cartridges C18 (standard density), 7 mm inner diameter, 3 mL volumes, Sigma]. Each fraction was concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1% formic acid for liquid chromatography-tandem mass spectrometry (LC-MS/MS).