Identification of Differentially Expressed Proteins Using iTRAQ Labeling

Because aspirin inhibits biofilm formation, we performed proteome analysis to obtain further information about proteins down- and up-regulated in the presence of aspirin. To identify differentially expressed proteins, biofilms treated with aspirin or untreated were labeled with isobaric reagents; the samples were pooled, fractionated by SCX chromatography, separated by LC and analyzed by MS/MS. Based on a fold-change of >1.2 or <0.8 (P-value < 0.05), 1,762 proteins were identified. We screened 178 differentially expressed proteins following biofilm formation under 1/2 MIC aspirin, of which 111 and 67 proteins were down- and up-regulated, respectively. Detailed information can be found in Supplementary Table S1. Meanwhile, raw data can be found in Supplementary Tables S2, S3, S4. In order to verify the accuracy of the data, the selected differential proteins were validated at the mRNA level by qPCR analysis, see Supplementary Figure S1. Given the inhibitory effects of aspirin on S. xylosus biofilms, special attention was given to proteins that demonstrated significant down-regulation of biofilm proteome in the presence of aspirin.