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Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
Published on: March 24, 2012
Identification of thioredoxin disulfide targets using a quantitative proteomics approach based on isotope-coded
Per Hägglund1, Jakob Bunkenborg, Kenji Maeda
1Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
Journal of Proteome Research
|April 16, 2009
Summary
This study introduces a new proteomics method to quantify thioredoxin (Trx) targets by measuring disulfide reduction. The method identified 104 Trx targets, revealing its role in translation and the ascorbate-glutathione cycle.
Area of Science:
- Biochemistry
- Proteomics
- Redox Biology
Background:
- Thioredoxin (Trx) is a key protein disulfide reductase involved in cellular redox homeostasis.
- Previous proteomics studies identified many Trx targets, but lacked molecular-level specificity due to unquantified reactivity.
- Understanding Trx target specificity is crucial for elucidating its diverse cellular roles.
Purpose of the Study:
- To develop and validate a novel proteomics procedure for quantifying Trx-mediated disulfide reduction.
- To identify specific protein disulfide targets of Trx in barley embryos.
- To gain molecular insight into Trx target specificity and its functional implications.
Main Methods:
- Developed a quantitative proteomics approach using isotope-coded affinity tag (ICAT) reagents for differential thiol labeling.
- Applied the method to barley embryo protein extracts treated with or without Trx.
- Quantified Trx-mediated disulfide reduction by measuring ratios of labeled tryptic peptides using LC-MS.
Main Results:
- Identified 104 significant Trx-reduced disulfide targets among 199 ICAT-labeled peptides.
- Confirmed known Trx targets like peroxiredoxin and cyclophilin.
- Discovered new targets, including ribosomal proteins, suggesting a link between Trx and translation.
- Dehydroascorbate reductase's catalytic cysteine was the most reduced target, highlighting Trx's role in the ascorbate-glutathione cycle.
Conclusions:
- The novel ICAT-based proteomics method enables precise quantification of Trx-mediated disulfide reduction.
- Thioredoxin targets a broad range of proteins, including those involved in translation and the ascorbate-glutathione cycle.
- These findings provide molecular-level insights into Trx specificity and expand our understanding of its cellular functions.
