Proteomics analysis of tongue in rats

The tongue samples were homogenized in 0.5 mL RIPA buffer for 4 min and then were staved with ultrasonic for 2 min in an ice bath. The lysates were centrifuged at 12000 rpm for 10 min at 4°C, and the supernatants were collected. Quantitative analysis of protein was performed by the BCA method. The distilled water was added into 100 μg protein extracts of each sample with a final concentration of 1 mg/mL. The pre-chill acetone (-20°C, 5 times volume of the sample) was added to the sample, mixed and then precipitated overnight at -20°C. The mixture was centrifuged at 12,000 rpm at 4 °C for 10 min, and then removed supernatant. Each sample was co-incubated with 5 mL of dithiothreitol (DTT) (200 mM) for 1 h at 55 C, followed by 15 min for the room temperature co-incubation of iodoacetamide (IAA, 10 mM). Samples were then digested by trypsin (Promega, Madison, WI, USA) at 50∶1 mass ratio (protein∶trypsin) overnight. The digested peptide product was labeled using TMT kits. Subsequently, peptides were desalted by C18 disks (Sigma­Aldrich, St. Louis, MO, USA) and vacuum-dried and separated using Waters XBridge BEH C18 XP Column (150 mm × 2.1 mm) to obtain high­PH fractionation peptides.