PEGS: SIMPLE AND AUTOMATABLE SAMPLE PREPARATION FOR TRYPTIC PEPTIDE MAPPING WITH THE PEPTIDEWORKS KIT

Importance of the topic

Therapeutic protein analysis demands robust and reproducible sample preparation workflows to ensure accurate peptide mapping. Efficient digestion minimizes method induced variations, supports regulatory compliance and enhances data reliability.

Study Objectives and Overview

This study evaluates a streamlined sample preparation workflow using Waters PeptideWorks Kit centered on RapiZyme Trypsin. The protocol is designed for both manual and automated operation on an Andrew plus Pipetting Robot to achieve rapid, complete tryptic digestion of proteins.

Methodology and Instrumentation

The workflow integrates:

• Denaturation, reduction and alkylation steps followed by neutralization and trypsin digestion
• RapiZyme Trypsin providing high activity, low autolysis and consistent performance
• Automation on Andrew plus Pipetting Robot for 24 samples in parallel within 2.5 hours

Instrumentation:

• ACQUITY UPLC I Class PLUS system with CSH C18 columns for peptide separation
• ACQUITY RDa and Xevo G2 XS mass spectrometers for qualitative and quantitative analysis

Main Results and Discussion

Both manual and automated digests of NIST monoclonal antibody showed over 88 percent sequence coverage with missed and non specific cleavages below five percent. Day to day variability remained under fifteen percent for unmodified peptides and under ten percent for modifications such as deamidation and oxidation. In ADC peptide mapping of a Trastuzumab biosimilar and Trastuzumab emtansine, additional drug conjugate related peaks and diastereomer pairs were identified without compromising digestion efficiency. Compared to a leading immobilized trypsin kit, the workflow achieved a ninety three percent reduction in missed cleavages and a fifty five percent reduction in non specific cleavages.

Benefits and Practical Applications

The approach offers:

• High digestion fidelity and minimal enzyme derived contaminants
• Scalable automation for high throughput sample processing
• Robust performance suitable for routine QA QC and advanced ADC characterization

Future Trends and Applications

Further integration with laboratory automation and AI driven data analysis is anticipated. The workflow may extend to multisite deployment, support other proteases and enhance speed through microfluidic and high pressure platforms.

Conclusion

The PeptideWorks Kit provides a comprehensive, automatable solution for rapid and reproducible tryptic peptide mapping. It enables high sequence coverage, low variability and significant reduction of cleavage artifacts, meeting the demands of therapeutic protein characterization.

References

No external references were provided in the source text.