An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Science

Background:

• Protein deamidation alters therapeutic protein characteristics and function.
• Cobratide, an analgesic, is susceptible to deamidation at the N48 residue, impacting safety and efficacy.
• Existing deamidation quantification methods lack efficiency and accuracy.

Purpose of the Study:

• To develop an improved 18O-labeling method for precise quantification of cobratide deamidation.
• To establish a robust quality control method for cobratide drug products.

Main Methods:

• Utilized an 18O-labeling strategy combined with Orbitrap high-resolution mass spectrometry.
• Detected a unique peptide containing the N48 deamidation hotspot after enzymolysis.
• Validated accuracy using synthesized peptides and capillary electrophoresis comparison.

Main Results:

• Achieved limits of detection and quantification of 0.02 and 0.025 μM, respectively.
• Demonstrated inter- and intra-day precision <3% and high accuracy (recoveries >101%).
• Confirmed method robustness and applicability for protein deamidation detection.

Conclusions:

• The developed 18O-labeling method provides accurate and efficient quantification of cobratide deamidation.
• This technique enhances quality control for protein therapeutics, ensuring drug safety and consistency.
• The study lays a foundation for future protein therapeutics research and drug quality evaluations.