Isotope-coded affinity tag approach to identify and quantify oxidant-sensitive protein thiols

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Protein thiols, particularly cysteine residues, are crucial for cellular function.
• Oxidation of protein thiols can lead to altered protein activity and cellular dysfunction.
• Accurate methods for quantifying thiol redox states are essential for understanding cellular signaling and disease.

Purpose of the Study:

• To develop and validate a novel approach for identifying and quantifying oxidant-sensitive protein thiols.
• To demonstrate the utility of isotope-coded affinity tag (ICAT) reagents in conjunction with mass spectrometry for thiol redox state analysis.
• To establish a proof-of-principle for applying this method in proteomics studies.

Main Methods:

• Utilized a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent for labeling free thiols.
• Employed mass spectrometry to quantify the relative abundance of ICAT-labeled peptides.
• Validated the approach using creatine kinase, a model protein with a known oxidant-sensitive cysteine residue.

Main Results:

• The ICAT method successfully identified and quantified changes in free thiols upon oxidative stress.
• Hydrogen peroxide treatment led to a decrease in the relative abundance of the unmodified thiol at cysteine-283 in creatine kinase.
• This confirmed the method's ability to detect oxidation of specific cysteine thiols.

Conclusions:

• The ICAT-based quantitative mass spectrometry approach is effective for identifying and quantifying oxidation of cysteine thiols.
• This methodology provides a valuable tool for proteomics research focused on protein thiol redox states.
• The approach facilitates deeper insights into cellular redox signaling pathways and their involvement in disease.