Protein Extraction, Digestion and Labeling With iTRAQ Reagents

Protein expression profile analysis was conducted for three samples which were exponential-phase cells (C sample), VBNC cells (V sample) and resuscitation cells (R sample). The cells were washed twice with cooled phosphate-buffered saline (PBS) and were centrifuged at 8,000 rpm for 5 min at 4°C. The collected cells were immediately stored at −80°C. Three biological replicates were used per sample. The bacterial cells were mixed with lysis solution (1% SDS, 200 mM DTT, protease inhibitor cocktail at a volume ratio of 1:100, 50 mM Tris–HCl, pH 8.8) at a ratio of 1:10, and incubated in ice-bath for 30 min, vortex-oscillated for 10 s every 10 min, then incubated at 100°C for 5 min. The mixture was centrifuged at 12,000 g, 4°C for 20 min. The supernatant was added with pre-cooled acetone at a ratio of 1:4. The proteins were precipitated overnight and centrifuged at 12,000 × g, 4°C for 20 min. The protein precipitate was mixed with 90% acetone for 10 s and then centrifuged at 12,000 × g, 4°C for 20 min. The protein precipitate was dissolved in the protein lysis solution (1% SDS, 8 M urea, protease inhibitor cocktail at a volume ratio of 1:100), and the supernatant was obtained by centrifugation at 4°C for 30 min. The protein concentrations were determined using a BCA Assay Kit (Thermo Fisher Scientific, United States). The sample solution was added with TCEP at the final concentration of 10 mM, and incubated at 37°C for 60 min. Then iodoacetamide was added (40 mM), the reaction took place in the dark at room temperature for 40 min. The solution was mixed with pre-cooled acetone at a volume ratio of 1:6, and put at −20°C for 4 h. The precipitate was collected by centrifugation at 10,000 × g for 20 min, dissolved with 100 μL of 100 mM TEAB, and then digested with trypsin at the mass ratio of 25:1 overnight at 37°C. The peptide mixture was labeled with the 8-plex iTRAQ reagent according to the protocol provided by the manufacturer (AB Sciex, United States).