Peptide Mapping of Ovalbumin Using Reversed- Phase High-Performance Liquid Chromatography and Prediction of Phosphopeptide Elution

Significance of the Topic

Protein phosphorylation and other post-translational modifications critically influence therapeutic protein activity, stability, and immunogenicity.
Peptide mapping offers single-amino-acid resolution to detect these modifications and ensure product identity and quality.
This method underpins biopharmaceutical development, quality control, and regulatory compliance.

Study Objectives and Overview

This application outlines a comprehensive workflow for mapping ovalbumin peptides, focusing on the identification of phosphopeptides.
Key aims include efficient reduction, alkylation, tryptic digestion, reversed-phase HPLC separation, and phosphopeptide confirmation by enzymatic dephosphorylation.

Used Instrumentation

• Dionex UltiMate 3000 HPLC system: SRD-3600 solvent rack with degassers, DGP 3600M pump, WPS-3000T autosampler, and PDA-3000 detector
• Acclaim 300 C18 analytical (2.1 × 150 mm) and guard (2 × 10 mm) columns (300 Å, 3 µm silica)
• Thermo Scientific SpeedVac concentrator (SVC100) with RVT400 vapor trap
• Standard lab equipment: microcentrifuge, Reacti-Therm heating block, dialysis tubing, injection vials

Methodology

Reduction and Alkylation
Proteins were denatured in 7 M guanidine HCl, reduced with DTT, and alkylated with iodoacetamide to stabilize disulfide bonds.
Tryptic Digestion
Sequencing-grade modified trypsin (1:50 w/w) was used; digestion proceeded at 37 °C for 20 h, followed by removal of buffer residues via SpeedVac.
HPLC Separation
Peptides were separated at 50 °C on the Acclaim 300 C18 column using a gradient from 5% to 80% acetonitrile in 0.1% TFA at 0.2 mL/min; detection at 214 nm.
Phosphatase Treatment
Aliquots of the digest were incubated with bovine intestinal alkaline phosphatase in Tris buffer (pH 9) to remove phosphates and reveal retention shifts.

Main Results and Discussion

System qualification with cytochrome C tryptic digest achieved retention time RSD ≤0.3% and peak area RSD ≤1.2%, confirming gradient and injection reproducibility.
Fetuin digests demonstrated high digestion consistency (36 peaks, peak area RSD ≤3.5%), validating the sample preparation protocol.
Ovalbumin mapping produced 35 peaks (theoretical 34), accounting for phosphorylated and miscleaved forms.
Two phosphopeptides, EVVGS*AEAGVDAASVSEFFR and LPGFGDS*IEAQCGTSVNVHSSLR, were identified by their disappearance and elution shift after phosphatase treatment, corroborated by absorbance at 260 nm and hydrophobicity calculations.

Benefits and Practical Applications

• Reliable and reproducible peptide mapping workflow for PTM characterization
• Seamless integration with LC-MS for advanced proteomic analysis
• High-resolution separation supports QA/QC and regulatory requirements in biopharmaceutical production

Future Trends and Possibilities

Ultra-high-pressure LC and novel column chemistries will further improve resolution and throughput.
Enhanced MS data analysis and bioinformatics tools will automate PTM identification.
Machine learning models may predict peptide retention and PTM effects, guiding method development.

Conclusion

The presented method provides a robust approach for ovalbumin peptide mapping and phosphopeptide identification using the Acclaim 300 C18 column on an UltiMate 3000 system.
Its reproducibility and sensitivity make it suitable for routine biopharmaceutical analysis.

References

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