Analyzing Trace Quantities of Amino Acid Sequences Using a Protein Sequencer —Gradient System—

Importance of the Topic

Protein sequencing remains a crucial analytical tool in biochemistry and molecular biology. Reliable identification of N terminal amino acid sequences, even in trace protein samples, supports research on disease mechanisms, biomarker discovery and drug development. Modern advances in sample pretreatment allow isolation of low abundance proteins, creating demand for high sensitivity sequencing instruments.

Objectives and Scope of the Study

This whitepaper presents an application example of the PPSQ-50A Series protein sequencer gradient system. Key aims include demonstrating:

• The ability to detect and sequence amino acids in femtomole to picomole protein samples
• Automated prediction of sequence data via integrated software
• Sequencing of unregistered proteins not found in genome libraries

Methodology and Instrumentation

The study employed Edman degradation chemistry coupled with a gradient HPLC separation. Analytical conditions were set as follows:

• Column type Wakopak Wakosil PTH-GR S-PSQ 250 mm × 2.0 mm I.D.
• Mobile phase A PTH amino acids mobile phase A for gradient elution
• Mobile phase B PTH amino acids mobile phase B for gradient elution
• Gradient profile 0 to 0 over 0–4 minutes, ramp to 100 over 17–30 minutes, return to 0 at 30.01–45 minutes
• Flow rate 0.3 mL per minute at 35 °C
• Detection wavelength UV 269 nm with high sensitivity flow cell

Instrumentation:

• PPSQ-50A Series Protein Sequencer Gradient System
• SPD-M30A UV detector
• Polymeric glass fiber sample plates treated with polybrene

Main Results and Discussion

Analysis of a standard mixture of PTH amino acids at 500 fmol each produced clear peak patterns through 30 sequencing cycles, confirming system sensitivity. Sequencing of calmodulin at 2 pmol total sample yielded usable N-terminal chromatograms across successive cycles, demonstrating reliable detection even at picomole levels. Difference chromatograms revealed characteristic peak shifts attributable to specific PTH amino acids, enabling sequence assignment.

Benefits and Practical Applications

The PPSQ-50A system offers high confidence in N-terminal sequencing without proteolytic digestion. Key advantages include:

• Distinction between isobaric residues isoleucine and leucine
• Direct mapping of disulfide bond locations
• Automated software tools for sequence prediction
• Capability to analyze proteins absent from genomic databases

These features support applications in QA QC laboratories, proteomics research and novel biomarker identification.

Future Trends and Potential Applications

Ongoing improvements in detector sensitivity, microfluidic sample handling and data processing algorithms will further reduce required sample quantity. Integration with mass spectrometry and high throughput platforms may expand use in clinical diagnostics and large scale proteome mapping. Enhanced bioinformatics pipelines could automate de novo sequencing of complex protein mixtures.

Conclusion

The PPSQ-50A protein sequencer gradient system achieves reliable N-terminal amino acid sequencing from trace protein samples. Its sensitivity, ease of use and software support make it an indispensable tool for modern protein analysis workflows.