Identification of thioredoxin target disulfides using isotope-coded affinity tags.

Area of Science:

• Biochemistry
• Proteomics
• Cellular Redox Biology

Background:

• Thioredoxins (Trx) are crucial small redox proteins involved in maintaining cellular thiol redox homeostasis.
• They function by reducing disulfide bonds in target proteins, playing a vital role in various cellular processes.
• Understanding Trx targets and their redox state is essential for comprehending cellular regulation.

Purpose of the Study:

• To develop and describe a novel thiol-specific labeling and affinity enrichment method.
• To enable the identification and relative quantification of thioredoxin (Trx) target disulfides in complex protein extracts.
• To provide a tool for monitoring the impact of redox agents on the thiol/disulfide proteome.

Main Methods:

• Utilized isotope-coded affinity tag (ICAT) reagents with thiol-reactive iodoacetamide and biotin affinity tags.
• Targeted peptides containing reduced cysteine residues for labeling.
• Employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of light ((12)C) and heavy ((13)C) labeled peptides.

Main Results:

• Successfully identified substrates for thioredoxins (Trx) in complex protein mixtures.
• Quantified the extent of target disulfide bond reduction mediated by Trx.
• Demonstrated the utility of the method for analyzing redox-sensitive proteins.

Conclusions:

• The described thiol-specific labeling and affinity enrichment approach is effective for identifying Trx targets.
• This methodology allows for the relative quantification of disulfide bond reduction, providing insights into redox control.
• The technique is adaptable for studying the effects of various redox agents on cellular thiol/disulfide proteomes.