Analysis of Long-Chain Amino Acid Sequences Using a Protein Sequencer — Gradient System —

Significance of the topic

The analysis of protein amino acid sequences is fundamental for understanding protein function, process optimization, and novel protein characterization in biochemistry and pharmaceutical research. Edman degradation remains a gold standard for N-terminal sequencing, offering direct amino acid identification for purified proteins and peptides.

Objectives and Overview of the Study

This study focuses on evaluating the performance of the PPSQ-50A gradient system protein sequencer for long-chain N-terminal amino acid sequence analysis. The objectives include improving peak resolution and sensitivity, reducing baseline noise, and demonstrating sequence identification on a mouse IgG heavy chain.

Methodology

• Edman Degradation: Sequential cleavage of N-terminal amino acids to form PTH derivatives.
• Gradient Elution: Use of mobile phases A and B to enhance separation of PTH-amino acids, particularly hydrophobic residues.
• Sample Preparation: SDS-PAGE of mouse serum IgG, electroblot onto PVDF membrane, CBB staining and excision of heavy chain bands.
• Baseline Optimization: Replacement of trimethylamine with N-methylpiperidine and adjustment of trifluoroacetic acid and dithiothreitol concentrations to minimize Edman by-products.
• Data Analysis: Software-assisted peak recognition based on intensity changes across sequencing cycles.

Instrumentation Used

• PPSQ-50A Protein Sequencer with gradient elution configuration.
• Column: Wakopak Wakosil PTH-GR, 250 mm × 2.0 mm I.D.
• Detector: SPD-M30A UV at 269 nm with high-sensitivity flow cell.
• Electrophoresis: SDS-PAGE 4–12% Bis-Tris gels.
• Membrane: PVDF for protein transfer and CBB staining.

Main Results and Discussion

The gradient system delivered PTH-amino acid peak intensities three to five times higher than isocratic methods, with improved peak shapes and lower background. By-product generation was significantly reduced, enhancing baseline stability. A 46-residue sequence from the mouse IgG heavy chain was successfully identified from 30 pmol sample quantities. The system demonstrated high sensitivity, enabling detection of trace-level sequences.

Benefits and Practical Applications

• Reliable N-terminal sequencing of purified proteins and peptides.
• Identification of sequences absent from genome databases.
• Complementary data to mass spectrometry for comprehensive protein characterization.
• Automated, user-friendly operation requiring minimal manual intervention.

Future Trends and Potential Applications

Advancements may include integration with mass spectrometric techniques, increased throughput through microfluidic platforms, and AI-driven sequence prediction. Continued reagent optimization could further suppress by-product formation and expand applicability to more challenging protein samples.

Conclusion

The PPSQ-50A gradient system enhances N-terminal amino acid sequencing by improving sensitivity, resolution, and operational ease. Its ability to identify extended sequences from trace samples underscores its value as a complementary tool to mass spectrometry in modern proteomic workflows.

References

• Kuriki T. Analysis of Long-Chain Amino Acid Sequences Using a Protein Sequencer — Gradient System. Shimadzu Application News, No. 01-00549-EN, April 2023.
• Shimadzu Corporation. Application News No. 01-00521.