Protein Extraction and Trypsin Digestion

The cell pellets were lysed in 0.4 ml lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2% SDS) followed by 10 times of a 10 s pulse sonication with 10 s rest between each time (Fisher Scientific Sonic Dismembrator Model 500, 15% amplitude). The lysate was centrifuged at 15,000 × g for 10 min at 4°C. Supernatants were collected and stored at −80°C for further analysis. The protein concentration of the supernatants was determined by a BCA^TM Reducing Reagent compatible assay kit (Thermo Scientific, Rockford, IL, United States). For trypsin digestion, an aliquot of 150 μg proteins were reduced with DTT followed by alkylation with iodoacetamide for 30 min at RT in the dark. Alkylated proteins were desalted on Amicon Ultra Centrifuge Filter Units (EMD Millipore) followed by trypsin digestion at 37°C overnight (16 h). After trypsin digestion, the samples were dried and stored at −80°C.