Here, we describe a procedure for the identification of S-nitrosothiols that has been used in our laboratory to study the roles of protein S-nitrosylation in the immune responses of Arabidopsis thaliana and other organisms. It employs a modified version of the biotin-switch technique, which we termed the sequential cysteine blocking technique, encompassing the sequential redox-blocking of recombinant proteins followed by LC-MS/MS analysis.
Keywords: Iodoacetamide; LC–MS/MS; Label-free quantitation; Redox-based posttranslational modification; S-nitrosothiols; S-nitrosylation; Sequential cysteine blocking.