2.3. Protein Extraction, Digestion, and Labeling with iTRAQ Reagents

Bacterial cells were resuspended in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5) containing 1 mM PMSF, 2 mM EDTA, and 10 mM DTT. The samples were then sonicated for further protein solubilization. After centrifugation (25,000×g, 4°C, 20 min), the protein concentration of the supernatants was determined using the Bradford assay. Each sample (100 μg) was digested with trypsin (Promega, Madison, WI, USA) at 37°C for 12 h. The three lysate replicates of M. hyopneumoniae strain 168 were labeled with iTRAQ reagents 113, 114, and 115, and of strain 168L with iTRAQ reagents 116, 117, and 119.