Identification of thioredoxin targets in guard cell enriched epidermal peels using cysTMT proteomics

Thioredoxins (Trx) play central roles in cellular redox regulation. Although hundreds of Trx targets have been identified using different approaches, the capture of targets in a quantitative and efficient manner is challenging. Here we report a high-throughput method using cysteine reactive tandem mass tag (cysTMT) labeling followed by liquid chromatography (LC)-mass spectrometry (MS) to screen for Trx targets. Compared to existing methods, this approach allows for i) three replicates of pairwise comparison in a single LC-MS run to reduce run-to-run variation; ii) efficient enrichment of cysteine-containing peptides that requires low protein input; and iii) accurate quantification of the cysteine redox status and localization of the Trx targeted cysteine residues. Application of this method in guard cell-enriched epidermal peels from Brassica napus revealed 80 Trx h targets involved in a broad range of processes, including photosynthesis, stress response, metabolism and cell signaling. The adaption of this protocol in other systems will greatly improve our understanding of the Trx function in regulating cellular redox homeostasis.

Redox homeostasis is tightly regulated for proper cellular activities. Specific protein-protein interactions between redox active molecules such as thioredoxin (Trx) and target proteins constitute the basis for redox-regulated biological processes. The use of cysTMT quantitative proteomics for studying Trx reactions enabled identification of potential Trx targets that provide important insights into the redox regulation in guard cells, a specialized plant cell type responsible for sensing of environmental signals, gas exchange and plant productivity.