Figure 1: Workflow of OxNSIL-based quantitative thiol redox proteomics The water (control) and the H2O2-treated Arabidopsis plants were grown on 14N (blue symbols)- and 15N (red symbols)-coded salt media. The total cellular proteins, containing both reduced form (-SH) and reversibly oxidized form (eg. -S-S-) from both control and treated plants, were mixed together in the Forward experiment at a ratio of 1 to 1. The biotin-tagged alkylating reagents, MPB and BIAM, were used to chemically label the reduced and the reversibly oxidized protein cysteine moiety, respectively. In between the tandem alkylation reactions, a reducing reagent TCEP was employed to reduce the reversibly oxidized thiol group to a reduced form. Biotin-tagged alkylation reagents-labeled peptides were purified through streptavidin affinity isolation and subjected to tandem LC-MS/MS analysis. The workflow was performed again in a Reciprocal experiment, in which the light (14N) isotope and heavy (15N) nitrogen isotope was used to label the H2O2-treated and the control plants, respectively.