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).