Systematic Fe(II)-EDTA Method of Dose-Dependent Hydroxyl Radical Generation for Protein Oxidative Footprinting

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Biology

Background:

• Understanding protein structure-function relationships is vital for cellular mechanisms.
• Existing methods for protein structural analysis can be complex or require specialized equipment.

Purpose of the Study:

• To present a quantitative, accessible method for protein oxidative footprinting.
• To enable high-resolution mapping of protein structures using common laboratory tools.

Main Methods:

• Hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry.
• Quantitative analysis of protein oxidation using mass spectrometry.
• Application to model proteins (lysozyme) and protein complexes (RAS-monobody, NIST mAb, PRC2).

Main Results:

• Achieved robust, reproducible, dose-dependent protein oxidation with single residue resolution.
• Successfully mapped protein interfaces and surfaces using the developed method.
• Demonstrated the method's efficacy on diverse protein systems.

Conclusions:

• The described Fenton chemistry-based method offers an accessible and cost-effective approach to protein oxidative footprinting.
• This technique provides high-resolution structural insights crucial for understanding protein function and disease.
• The method is readily implementable in standard research and process control laboratories.