Preparation of protein extraction, separation of proteins and in-gel trypsin digestion

Total protein extraction from cell pellets was prepared by the following method. In brief, cell pellets were lysed in 0.4 ml lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitor cocktail pill). After cells were lysed, 50 μl of 10% SDS and 50 μl of 1 M DTT were added into the mixture followed by incubation at 95°C for 10 min. The extraction was then sonicated and centrifuged at 15,000 × g for 10 min. Supernatants were collected and stored at −80°C for further analysis. The protein concentration of the supernatants was determined by a BCA™ reducing reagent compatible assay kit (Thermo Fisher Scientific, Rockford, IL, USA).

Equal amounts of protein (130 μg) from each sample were fractioned by separation on a NuPAGE 4–12% Bis-Tris Gel (Life Technologies, Grand Island, NY, USA). Sixteen gel fractions from each lane representing one sample were treated with DTT for reduction, then iodoacetamide for alkylation, and further digested by trypsin in 25 mM NH₄HCO₃ solution. The digested protein was extracted, and the extracted peptides were dried and reconstituted in 20 μl of 0.1% formic acid before nanospray LC/MS/MS analysis was performed.