Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry

Area of Science:

• Biochemistry
• Pharmaceutical Science
• Analytical Chemistry

Background:

• Protein therapeutics are crucial in modern medicine.
• Existing methods struggle with rapid, high-resolution analysis of protein higher-order structures.
• Ensuring the efficacy and safety of protein drugs requires detailed structural characterization.

Purpose of the Study:

• To develop a rapid, accurate, and reproducible method for characterizing three-dimensional structural changes in protein therapeutics.
• To identify specific protein regions involved in aggregation and structural alterations due to mishandling.
• To assess the utility of diethylpyrocarbonate (DEPC) labeling coupled with mass spectrometry for this purpose.

Main Methods:

• Utilized diethylpyrocarbonate (DEPC) labeling combined with mass spectrometry.
• Applied the method to model protein systems: β2-microglobulin, immunoglobulin G1, and human growth hormone.
• Analyzed DEPC-labeled proteins to detect structural modifications.

Main Results:

• DEPC labeling successfully identified protein regions mediating aggregation.
• The method detected subtle structural changes in proteins exposed to degrading conditions.
• Information was obtained for up to 30% of surface residues, indicating high structural resolution.
• Demonstrated applicability to diverse protein therapeutics and formulations.

Conclusions:

• DEPC labeling coupled with mass spectrometry offers a simple and effective approach for analyzing protein therapeutic structures.
• This method provides high-resolution insights into structural changes, crucial for drug quality control.
• The technique is adaptable for various protein therapeutics, enhancing drug safety and efficacy assessment.