2.7. Spot picking and in-gel digestion

A total of 1 mg of unlabeled proteins was used to run 2-DE using identical conditions as described above and stained with coomassie brilliant blue G-250 staining solution. The significant different protein spots detected by Decyder 2D v6.5 software analysis were manually excised by Coomassie Blue G-250 staining gel. The gel pieces were destained with 50% acetonitrile in 25 mM ammonium bicarbonate and then dehydrated in 100% acetonitrile. After the reagents were removed, each gel piece was digested overnight at 37°C with 0.01 mg/μL of sequencing grade trypsin (Promega, USA) in 15 μL digestion buffer containing 25 mM ammonium bicarbonate.