Covalent Labeling Denaturation Mass Spectrometry for Sensitive Localized Higher Order Structure Comparisons

Area of Science:

• Biochemistry
• Analytical Chemistry
• Protein Science

Background:

• Protein higher order structure (HOS) is crucial for protein function and a key attribute in biopharmaceutical development.
• Covalent labeling coupled with mass spectrometry is used to assess localized protein HOS.
• Conventional methods often use minimal labeling to preserve protein structure.

Purpose of the Study:

• To present a new method using high concentrations of isobaric labels to detect localized protein HOS differences.
• To enhance sensitivity and resolution for identifying structural variations in biological products.
• To demonstrate the method's ability to quantify other product attributes.

Main Methods:

• Utilizing high amounts of isobaric labels, sufficient to induce denaturation, for enhanced HOS analysis.
• Employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for data acquisition.
• Implementing automated data analysis for efficient processing.

Main Results:

• The method detected localized HOS differences at levels as low as 2.5-5% of the molecular population.
• Demonstrated higher sensitivity compared to circular dichroism and nuclear magnetic resonance spectroscopy fingerprinting for specific HOS variations.
• Showed comparable sensitivity to differential scanning calorimetry for detecting HOS differences.
• Successfully quantified post-translational modifications and site-specific glycosylation in a single LC-MS/MS run.

Conclusions:

• High-labeling mass spectrometry is a highly discriminative technique for detecting subtle protein HOS variations.
• This workflow offers a sensitive and comprehensive approach for biopharmaceutical characterization.
• The method is applicable to large, multiply glycosylated therapeutic proteins under various stress conditions.