Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Reactive oxygen species (ROS) are vital signaling molecules, but their abnormal levels contribute to diseases like cancer.
• Understanding protein oxidation is key to deciphering reduction-oxidation (redox) signaling pathways.
• Specific protein oxidation changes are implicated in hereditary leiomyomatosis and renal cell carcinoma (HLRCC).

Purpose of the Study:

• To develop and validate a novel proteomic approach for quantifying reversible protein oxidation.
• To apply this method to investigate protein oxidation in hereditary leiomyomatosis and renal cell carcinoma (HLRCC).

Main Methods:

• Developed an Activated Thiol Sepharose-based proteomic (ATSP) approach.
• ATSP enriches for H2O2-sensitive thiol peptides containing reactive cysteines.
• Applied ATSP to analyze protein oxidation in FH-deficient HLRCC cells and cells treated with specific compounds or growth factors.

Main Results:

• Identified multiple oxidized proteins in FH-deficient HLRCC cells, including metabolic proteins like pyruvate kinase M2 (PKM2).
• Observed changes in PKM2 oxidation upon treatment with dimethyl fumarate or PKM2 activators.
• Successfully detected oxidation of specific proteins (e.g., Src homology region 2 domain-containing phosphatase-2, PKM2) in stimulated cells.

Conclusions:

• The ATSP workflow effectively quantifies reversible cysteine oxidation in proteins.
• This redox proteomics method is applicable to various physiological and pathological conditions, including HLRCC.
• The findings provide insights into redox signaling dysregulation in kidney cancer.