Fig. 1: Development of FAT-switch approach for the detection of S-nitrosoproteome in Arabidopsis.

a Reaction schema for FAT-switch approach to label S-nitrosylated proteins and enrich target peptides. Step 1, free cysteine thiols were blocked by IAM. In step 2, the S-nitrosylated cysteines were specifically reduced by a low concentration of ascorbate and substituted by a sulfhydryl reactive fluorous tag reagent. Step 3, proteins were digested by trypsin. After then, C18 column were used to capture fluorous-tag labeling peptides and remove high hydrophilic peptide by a low ACN concentration of washing buffer (Step 4). Finally, fluorous labeled peptides were further purified by nano-graphite fluoride, and identified by LC-MS/MS (Step 5 and 6). b The number of S-nitrosylation peptides identified by biotin switch method and FAT-switch approach with same bench of triplicate samples. Error bars, SD (n = 3 biological replicates). c Boxplots of log₁₀ MS/MS intensity of all identified S-nitrosylation peptides in biotinswitch method and FAT-switch approach with same bench of triplicate samples. d Number of S-nitrosylation peptides identified per μg of Arabidopsis proteins. Error bars, SD (n = 3 biological replicates).