Non-Reducing Proteomics Reveals Disulfide-dependent Proteoform Remodeling Under Oxidative Stress

Area of Science:

• Biochemistry
• Cell Biology
• Proteomics

Background:

• Oxidative stress induces post-translational modifications, including disulfide bonds.
• Disulfide bonds are often missed in proteomics due to reducing agents.
• Hydrogen peroxide (H2O2) is a key mediator of oxidative stress.

Purpose of the Study:

• To investigate the effects of H2O2 on MDA-MB-231 cells using metabolomics and non-reducing proteomics.
• To identify and characterize disulfide bond formation in proteins under oxidative stress.
• To explore the role of disulfide bonds in redox regulation.

Main Methods:

• Liquid chromatography-mass spectrometry (LC-MS) based metabolomics.
• Non-reducing tandem mass tag (TMT) proteomics with the DBond algorithm.
• Analysis of H2O2-treated MDA-MB-231 cells.

Main Results:

• Metabolomic analysis showed pathway-specific inhibition of glycolysis, TCA cycle, and nucleotide biosynthesis.
• Proteomic analysis identified over 1,000 proteins with disulfide crosslinks.
• Disulfide linkages were enriched at redox-sensitive cysteines and showed high isoform specificity.

Conclusions:

• Disulfide bond formation is a selective mechanism for redox regulation.
• Non-reducing proteomics is valuable for studying redox-controlled protein networks.
• This study provides insights into cellular structural dynamics under oxidative stress.