2.2 Protein extraction, digestion, and TMT labelling
1% PVPP and appropriate amount of BPP solution were added to the samples, and then agitated 3 times for 40 s each using a high-throughput tissue grinder. The supernatant was centrifuged at 1200 g for 20 min followed by tris-aturated phenol and BPP solution added and vortexed for 10 min at 4 ℃. Protein was precipitated overnight at –20 °C in 5 times volumes of pre-chilled ammonium acetate methanol solution, and the following day it was centrifuged at 12000 g for 20 min at 4 °C. The precipitate was mixed with 90% pre-cooled acetone, and the supernatant was discarded and repeated twice. The obtained precipitate was dissolved with protein lysis solution. The protein concentrations were determined by the BCA method [24].
100 μg of protein from each sample was diluted with an appropriate amount of TEBA. Following the addition of TCEP, which was allowed to react at 37 °C for 60 min, 1AM was added and allowed to react at room temperature for 40 min. The alkylated proteins were precipitated by adding six times the volume of pre-cooled acetone at a temperature of –20 °C for 4 hours, followed by centrifugation at 10000 g for 20 min. 100 mL of 100 mM TEBA was used to completely dissolve the precipitate. In order to complete the enzymatic digestion, trypsin was added at a ratio of 1:50 (enzyme: protein) and left overnight at 37 °C.
TMT Reangent kit (Thermo, USA) was used to label the resultant peptides in accordance with the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 100 μg of peptide were mixed with a tube of TMT reagent and incubated at room temperature for 2 h. After mixed with hydroxylamine, the derivatization reaction was burst for 30 min. The TMT-labeled samples were then mixed and vacuum centrifuged dried. Six TMT-labeled samples were ultimately examined utilizing a Thermo Scientifir Vanquish F UHPLC system in combination with a Q-Exactive hybrid quadrupole orbital trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).