Fig. 1: Workflow of the biotin-switch SILAC method used to identify and quantify redox-modified protein thiols in E. coli biofilm and planktonic conditions.

A The biotin-switch method consists of the sequential blocking, reduction, and labeling of cysteine thiols; for the differential detection of S-NO and S-OX, cysteines are first reduced with ascorbate, a mild reducing agent, and then with the strong reducing agent DTT. Labeled peptides are then selectively eluted and collected for identification by mass spectrometry (LC-MS/MS). B Accurate peptide quantification using a SILAC approach was performed on each sample using heavy isotope lysine and arginine labeled samples as internal references. C E. coli proteins were extracted from four different cultures: planktonic shake flasks or biofilm microfermenters, each under aerobic or anaerobic conditions. Each condition included five biological replicates.