Improving the Yield of Basic Amino Acids in a Protein Sequencer

Importance of the Topic

Accurate determination of N-terminal amino acid sequences is crucial for quality control in biopharmaceutical manufacturing. Edman degradation remains a gold standard for sequencing, but low yields of basic amino acids (arginine, histidine, lysine) can compromise sequence interpretation, especially at trace sample levels. Enhancing these yields supports reliable verification of therapeutic proteins and antibodies in compliance with regulatory guidelines.

Objectives and Study Overview

This study aimed to develop and validate modifications to the Edman degradation workflow on the PPSQ-50A protein sequencer, with the goal of raising yields of basic PTH-amino acids. Bovine serum albumin (BSA) was used as a model protein to assess sequence cycles containing lysine, histidine, and arginine residues.

Methodology and Instrumentation

• PPSQ-50A Sequencer with PPSQ-51A/53A gradient system was applied for automated Edman cycles and gradient elution.
• Analytical separation employed a Wakopak Wakosil PTH-GR column (250 mm × 2.0 mm I.D.) with a gradient of PTH-amino acid mobile phases A and B at 0.3 mL/min, 35 °C, and UV detection at 269 nm.
• Standard mixture of 500 fmol PTH-amino acids validated baseline separation and detection sensitivity.
• BSA sequencing compared a standard protocol with an improved protocol incorporating two key changes: extraction of ATZ-amino acids under basic conditions and use of polybrene in sample application to stabilize basic side chains.

Main Results and Discussion

Improved protocol yielded substantially higher signals for PTH-lysine, PTH-histidine, and PTH-arginine in cycles 4, 9, 10, and 12 of BSA sequencing. Differential chromatogram analysis demonstrated increased peak heights for basic residues, with negligible interference despite a minor rise in background noise. These enhancements simplified peak identification and automated sequence assignment.

Benefits and Practical Applications

• Increased reliability of N-terminal sequencing for proteins containing basic residues, even at low sample loads.
• Streamlined data interpretation and automated software estimations due to improved peak clarity.
• Reduced risk of sequence misassignment in quality control protocols for biotherapeutic development.

Future Trends and Opportunities

• Integration of further chemical modifications or stabilizing agents to boost yields of other challenging residues.
• Application of the improved workflow to de novo sequencing of peptides and proteins with post-translational modifications.
• Coupling with mass spectrometric detection for comprehensive sequence verification and structural analysis.

Conclusion

The modified Edman degradation protocol on the PPSQ-50A platform significantly enhances the yield of basic PTH-amino acids without compromising sequence clarity. Adoption of these simple pretreatment and application steps offers a robust approach for accurate N-terminal sequencing in biopharmaceutical quality control.

References

• Shimadzu Corporation. PPSQ™-50A Series Protein Sequencer Application Note, First Edition April 2023.