Protein digestion and iTRAQ labeling

Protein digestion was performed according to the reported FASP procedure (Wisniewski et al., 2009). In brief, 200 μg of proteins at two different conditions (1/4 MIC of tylosin treated cells and nontreated cells) were added into 30 μL STD buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0) and ultrafiltered (Microcon units, 30 kD) with UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0). To block reduced cysteine residues, 100 μL 0.05 M iodoacetamide was added into UA buffer and incubated for 20 min in the dark. The filters were washed three times with 100 μL UA buffer and twice with 100 μL DS buffer (50 mM triethylammoniumbicarbonate at pH 8.5). Finally, the proteins were digested with 2 μg trypsin (Promega) in 40 μL DS buffer at 37°C for 16–18 h. Then, the resulting peptides were collected as a filtrate. The peptide content was estimated by UV light spectral density at 280 nm using an extinctions coefficient of 1.1 of 0.1% (g/l) solution calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins.

For the iTRAQ labeling, the peptides were labeled with the 8-plex iTRAQ reagent by following the manufacturer's instructions (Applied Biosystems). Each iTRAQ reagent was dissolved in 70 μL of ethanol and added to the respective peptide mixture. The peptides from the S. suis biofilms treated by tylosin were labeled with 115 isobaric reagent, and the peptides from the nontreated S. suis biofilms were labeled with 116 isobaric reagent. Then, the samples were multiplexed and vacuum dried. Three independent biological experiments were performed.