Structural Characterization of Multiple GLP-1 Receptor Agonists using Electron Capture Dissociation

Importance of the topic

The prevalence of type 2 diabetes and obesity has driven rapid growth in the development of GLP-1 receptor agonists. Early and accurate structural characterization of these therapeutic peptides is essential to ensure sequence fidelity, confirm post-translational modifications, and accelerate candidate selection in drug discovery.

Objectives and study overview

This study applies liquid chromatography coupled with tandem mass spectrometry using electron capture dissociation (ECD) to achieve in-depth sequence mapping and modification localization of three GLP-1 receptor agonists: semaglutide, liraglutide, and tirzepatide. The goal was to demonstrate comprehensive characterization, including isomer detection and modification retention, using Agilent instrumentation and ExDViewer software.

Methodology and instrumentation

• Sample preparation: Peptides at 10 μM in 15 % acetonitrile with 0.1 % formic acid; equimolar mixtures for LC analysis.
• Liquid chromatography: Agilent 1290 Infinity II Bio LC System with AdvanceBio Peptide Mapping column.
• Mass spectrometry: Agilent 6545XT AdvanceBio LC/Q-TOF equipped with ExD cell for ECD fragmentation; direct infusion for charge-state studies without internal calibration.
• Data analysis: ExDViewer software for targeted deconvolution, custom amino-acid and modification definitions, and automated fragmentation annotation.

Main results and discussion

• Intact mass determination: Monoisotopic masses measured as semaglutide 4 112.12 Da, liraglutide 3 749.95 Da, tirzepatide 4 811.53 Da, with isotopically resolved signals deconvoluted effectively by ExDViewer.
• Charge-state optimization: The 4+ precursor yielded 100 % sequence coverage with optimal fragment intensity; 3+ ions required supplemental collision energy for improved fragmentation, while 5+ ions risked over-fragmentation.
• Isoaspartate monitoring: ECD-specific c+57 and z–57 fragments facilitated detection of isoAsp in model peptides; analysis of liraglutide confirmed absence of isoAsp at Asp8 position.
• Modification localization: ECD preserved fatty-acid and non-standard amino-acid modifications (e.g., 2-aminoisobutyrate and Lys20 acylation in semaglutide), enabling precise mapping without extensive CID tuning.

Benefits and practical applications

This workflow delivers robust structural confirmation for synthetic peptide therapeutics, supporting quality control, regulatory submissions, and accelerating lead optimization by providing high-confidence sequence and modification data early in development.

Future trends and potential applications

• Integration with automated data processing and artificial intelligence for higher throughput and predictive fragmentation analysis.
• Extension to larger biopharmaceuticals and antibody-drug conjugates to map complex post-translational modifications.
• Combination with complementary fragmentation techniques (e.g., UVPD) to further enhance coverage and isomer discrimination.
• Application in personalized medicine for rapid verification of peptide variants and analogs.

Conclusion

Electron capture dissociation coupled with high-resolution LC/Q-TOF MS and ExDViewer software provides a comprehensive platform for detailed sequence mapping, modification localization, and isomer detection of GLP-1 receptor agonists, meeting critical needs in peptide drug development.

Reference

1. Comprehensive Characterization of Multiple GLP-1 Analogs Using an Agilent 6545XT AdvanceBio LC/Q-TOF with ECD and Agilent ExDViewer software. Agilent Technologies application note 5994-7994EN, 2025.
2. Identification of Amino Acid Isomers Using Electron Capture Dissociation in the Agilent 6545XT AdvanceBio LC/Q-TOF System. Agilent Technologies application note 5994-7506EN, 2024.