Streamlined Analysis of Synthetic Peptides with Non-Standard Amino Acid Chemistry

Importance of the Topic

Analyzing synthetic peptides that incorporate non-standard amino acids and backbone modifications is critical for the development of novel therapeutics, quality control in pharmaceutical manufacturing, and advancing proteomics research. Traditional fragment analysis tools often fail to accommodate these unique chemistries, leading to labor-intensive manual annotation and potential errors. Implementing a streamlined, automated workflow enhances data quality, speeds up characterization, and supports regulatory compliance in drug discovery and industrial analytics.

Goals and Overview of the Study

This work demonstrates a flexible approach to peptide fragmentation analysis that supports non-standard residues and peptoid backbones. Key objectives include:

• Customizing fragmentation rules to capture unique c/z-ion patterns in peptoids during electron capture dissociation (ECD).
• Validating the workflow on GLP-1 analogs (liraglutide, semaglutide, tirzepatide) and diverse peptoid samples.
• Highlighting automated annotation accuracy compared to manual curation.

Methodology and Instrumentation

The peptide samples were analyzed using an Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer equipped with an ExD cell for ECD and standard collision induced dissociation (CID) experiments. Data processing employed ExDViewer version 4.6.26 with the following features:

• Building block editor to define custom amino acids and peptoid monomers.
• Modification editor for specifying neutral losses and custom side-chain chemistries.
• Custom fragmentation rules for c and z ions tailored to non-standard backbone linkages.
• Caliper tool for manual de novo sequencing of unknown peptoid mixtures.

Main Results and Discussion

CID analysis of peptoids yielded expected b/y fragment patterns without additional rule customization, facilitating sequence mapping in both purified and crude samples. The caliper tool enabled de novo identification of up to 10 out of 11 consecutive peptoid monomers in complex mixtures. ECD of peptoids required bespoke c/z fragmentation definitions to account for side chains on backbone nitrogen, and automated annotation in ExDViewer closely matched manual assignments from literature datasets. For GLP-1 analogs, full sequence coverage was achieved, including golden ion pairs and triplets that enhanced confidence. ECD localization of a custom fatty-acid modification on tirzepatide was demonstrated by detecting consecutive fragment ions flanking the modified lysine.

Benefits and Practical Applications

The described workflow offers:

• Automated, high-confidence annotation of peptides with unusual chemistries, reducing manual effort.
• Comprehensive sequence coverage and modification mapping for biotherapeutics and research targets.
• Compatibility with regulatory QA/QC pipelines and accelerated characterization cycles.
• Adaptability to new building blocks and modifications via user-configurable editors.

Future Trends and Applications

Emerging directions include:

• Integration of machine learning to predict fragmentation patterns for novel chemistries.
• Expansion of standardized libraries of synthetic monomers for broader coverage.
• Real-time data processing and feedback in peptide manufacturing workflows.
• Cloud-based collaborative platforms for shared fragmentation rule sets and community-driven method development.

Conclusion

Agilent ExDViewer, combined with the Agilent 6545XT LC/Q-TOF system, provides a versatile, user-configurable platform for the automated analysis of synthetic peptides and peptoids. Custom fragmentation rules, building block and modification editors enable accurate, streamlined characterization of non-standard amino acids and backbone chemistries, supporting advanced research and industrial applications.

Reference

1. Bogdanov B, Zhao X, Robinson DB, Ren J. Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids. Journal of the American Society for Mass Spectrometry. 2014;25(7):1202–1216.
2. Agilent Technologies. Comprehensive Characterization of Multiple GLP-1 Analogs Using an Agilent 6545XT AdvanceBio LC/Q-TOF with ECD and Agilent ExDViewer Software. Application Note 5994-7994EN; 2025.
3. Agilent Technologies. Identification of Amino Acid Isomers Using Electron Capture Dissociation in the Agilent 6545XT AdvanceBio LC/Q-TOF System. Application Note 5994-7506EN; 2024.