title: Identification of thioredoxin targets in guard cell enriched epidermal peels using cysTMT proteomics.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL 32610, USA; Genetics Institute, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL 32610, USA; Genetics Institute, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Mengmeng
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL 32610, USA; Genetics Institute, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhu
authors.affiliation: Department of Chemical Engineering, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Strul
authors.affiliation: Thermo Fisher Scientific, 1400 Northpoint Parkway, West Palm Beach, FL 33407, USA.
authors.forename: Craig P
authors.initials: CP
authors.lastname: Dufresne
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL 32610, USA; Genetics Institute, University of Florida, Gainesville, FL 32610, USA; Department of Chemical Engineering, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Jacqueline D
authors.initials: JD
authors.lastname: Schneider
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL 32610, USA; Genetics Institute, University of Florida, Gainesville, FL 32610, USA; Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Alice C
authors.initials: AC
authors.lastname: Harmon
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL 32610, USA; Genetics Institute, University of Florida, Gainesville, FL 32610, USA; Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL 32610, USA; Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610, USA. Electronic address: schen@ufl.edu.
authors.forename: Sixue
authors.initials: S
authors.lastname: Chen
mesh_terms: D029688:Brassica napus / Q000378:metabolism*; D010084:Oxidation-Reduction; D019441:Plant Epidermis / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D013879:Thioredoxins
keywords: CysTMT; Epidermal peels; Guard cells; LC–MS/MS; Redox proteomics; Thioredoxin targets