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Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation
Published on: June 21, 2021
Comprehensive plasma thiol redox status determination for metabolomics.
Lisa A D'Agostino1, Karen P Lam, Richard Lee
1Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.
Journal of Proteome Research
|November 9, 2010
Summary
This study presents an advanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) method for precise plasma thiol analysis. The new technique enhances sensitivity and accuracy for assessing thiol homeostasis, crucial for understanding oxidative stress and aging.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Metabolomics
Background:
- Thiol homeostasis is vital for cellular redox balance and response to oxidative stress.
- Current methods for assessing plasma thiol/disulfide redox status face limitations in sensitivity, selectivity, and dynamic range for clinical research.
- Reliable assessment of thiol redox status is crucial for understanding its role in human health and aging.
Purpose of the Study:
- To introduce an improved, artifact-free strategy for comprehensive plasma thiol speciation using CE-ESI-MS.
- To overcome sensitivity, selectivity, and dynamic range limitations of conventional techniques for thiol analysis.
- To enable simultaneous analysis of reduced thiols and oxidized disulfides at nanomolar levels.
Main Methods:
- Development of an improved CE-ESI-MS strategy integrating specific and nonspecific approaches for sensitivity enhancement.
- Creation of a multivariate model to predict ionization efficiency for reduced thiols conjugated to maleimide analogs.
- Optimization of maleimide labeling and online sample preconcentration for simultaneous analysis.
- Utilizing CE migration time prediction as a complementary tool for metabolite identification.
Main Results:
- Achieved artifact-free quantification of labile plasma thiols with enhanced sensitivity and selectivity.
- Enabled simultaneous analysis of nanomolar levels of reduced thiols and free oxidized thiols (symmetric/mixed disulfides).
- Successfully identified low-abundance thiols and other polar metabolites in plasma samples.
- Demonstrated the capability for plasma thiol redox status determination and untargeted metabolite profiling.
Conclusions:
- The developed CE-ESI-MS method provides a robust and sensitive approach for comprehensive plasma thiol analysis.
- This technique overcomes limitations of conventional methods, facilitating reliable clinical research on thiol redox status.
- Combined thiol redox status determination and metabolite profiling offer a systemic strategy to investigate the role of dysregulated thiol metabolism in diseases.
