Thioredoxin-Dependent Decomposition of Protein S-Nitrosothiols

The addition of nitric oxide to cysteine moieties of proteins results in the formation of S-nitrosothiols (SNO) that have emerged as important posttranslational signaling cues in a wide variety of eukaryotic processes. While formation of protein-SNO is largely nonenzymatic, the conserved family of Thioredoxin (TRX) enzymes are capable of selectively reducing protein-SNO. Consequently, TRX enzymes are thought to provide reversibility and specificity to protein-SNO signaling networks. Here, we describe an in vitro methodology based on enzymatic oxidoreductase and biotin-switch techniques, allowing for the detection of protein-SNO targets of TRX enzymes. We show that this methodology identifies both global and specific protein-SNO targets of TRX in plant cell extracts.