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Peptide separations using fluorescence detection.

E H Creaser, G J Hughes

    Journal of Chromatography
    |November 21, 1977
    PubMed
    Summary
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    This study presents a sensitive method using o-phthalaldehyde to analyze peptides from protein digestion. The system efficiently separates and quantifies peptides, aiding in protein analysis and preparation.

    Area of Science:

    • Biochemistry
    • Analytical Chemistry
    • Proteomics

    Background:

    • Amino acids and peptides are crucial biological molecules.
    • Sensitive detection methods are needed for analyzing peptides from protein digestion.
    • O-phthalaldehyde (OPA) is a known reagent for fluorophore formation with primary amines.

    Purpose of the Study:

    • To describe a simple and sensitive system for the analysis and separation of peptides.
    • To enable peptide quantification in the nanomole to micromole range.
    • To facilitate the preparation of peptides derived from protein digestion.

    Main Methods:

    • Utilized o-phthalaldehyde (OPA) for sensitive fluorophore-based peptide detection.
    • Employed a cation-exchange chromatography system with volatile pyridine buffers for peptide separation.

    Related Experiment Videos

  • Integrated a gradient-generating device, pumps, and a fluorocolorimeter for real-time analysis.
  • Adapted the system for both analytical (detection) and preparative (collection) workflows.

    • Main Results:

    • Demonstrated a highly sensitive assay for amino acids and peptides using OPA.
    • Successfully analyzed and separated peptides in the 5 nmole to 10 micromole range.
    • Applied the system to analyze tryptic peptides from cytochrome c and Salmonella histidinol dehydrogenase.
    • Showcased the system's capability for both peptide analysis and preparative isolation.

    Conclusions:

    • The described system offers a robust and sensitive method for peptide analysis and separation.
    • This technique is valuable for studying peptides derived from enzymic protein digestion.
    • The OPA-based fluorometric assay provides excellent sensitivity for peptide quantification.