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

Importance of the Topic

The analysis of N-terminal amino acid sequences remains a cornerstone for protein characterization and quality control in research and biopharmaceutical production. Edman degradation-based sequencing enables high precision identification of residues at low sample quantities, supporting the development and assurance of protein therapeutics.

Objectives and Study Overview

This study evaluates the performance of the Shimadzu PPSQ-50A isocratic protein sequencer for long-chain N-terminal sequencing, including discrimination of isobaric residues, at picomole sensitivity.

Methodology

Proteins separated by electrophoresis are electroblotted onto PVDF membranes and dyed. Excised spots undergo Edman degradation cycles within the PPSQ-50A reactor. PTH-amino acids are released and analyzed by isocratic HPLC, using fixed elution conditions to ensure consistent retention times and reliable peak identification.

Used Instrumentation

• Shimadzu PPSQ-50A isocratic protein sequencer
• Wakopak Wakosil PTH-II column (250 mm × 4.6 mm I.D.)
• SPD-M30A UV detector at 269 nm with high-sensitivity flow cell
• Isocratic mobile phase recycling system
• PVDF membrane and electroblot apparatus

Main Results and Discussion

Baseline separation of a 25 pmol PTH-amino acid standard allowed unambiguous assignment of 19 common residues. The isocratic system maintained constant elution positions, simplifying cycle-to-cycle comparison. Analysis of 30 pmol erythropoietin enabled identification of up to the 49th N-terminal residue, demonstrating high sensitivity and effective differentiation of leucine and isoleucine.

Benefits and Practical Applications

• Reliable N-terminal sequence identification in purified proteins
• Automatic sequence prediction by dedicated software
• Trace-level analysis down to picomole amounts
• Cost-effective operation via mobile phase recycling
• Environmental advantages from reduced liquid waste
• Clear differentiation of isobaric residues

Future Trends and Applications

Integration with high-resolution mass spectrometry and advanced data analytics will enhance sequence coverage and throughput. Miniaturization and further automation promise faster workflows for complex proteins and post-translational modification mapping. Expansion into real-time process monitoring could benefit biopharmaceutical manufacturing and quality control.

Conclusion

The Shimadzu PPSQ-50A isocratic system delivers a robust, user-friendly platform for high-sensitivity N-terminal sequencing, addressing both research and quality assurance needs in protein science.

References

No external references cited.