University of Florida, USA
Tong Zhang received his PhD degree in 2016. His research has been focused on the application of proteomics and mass spectrometry in the field of plant cell signaling. For the past five years, he has conducted research in areas of kinase signaling and redox regulation. He also mentored students at different levels including high school, undergraduate and graduate students.
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; 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.