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Related Experiment Videos

HPLC shadowing: artifacts in peptide characterization monitored by RIA.

A J Fischman, A J Kastin, M V Graf

    Peptides
    |September 1, 1984
    PubMed
    Summary

    High performance liquid chromatography (HPLC) combined with radioimmunoassays (RIA) can cause false positives due to peptide carry-over, known as the shadow phenomenon. Researchers found this effect significant with delta sleep-inducing peptide (DSIP), highlighting the need for caution in peptide identification.

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    Area of Science:

    • Biochemistry
    • Analytical Chemistry
    • Pharmacology

    Background:

    • High-performance liquid chromatography (HPLC) is crucial for peptide characterization.
    • Radioimmunoassays (RIA) are sensitive detection methods often coupled with HPLC.
    • The 'shadow phenomenon' in HPLC-RIA can lead to misinterpretation of results.

    Purpose of the Study:

    • To investigate the occurrence and extent of the shadow phenomenon in HPLC-RIA systems.
    • To assess the impact of peptide carry-over on endogenous peptide detection.
    • To evaluate the reliability of HPLC-RIA for identifying peptides like delta sleep-inducing peptide (DSIP).

    Main Methods:

    • Utilized high-performance liquid chromatography (HPLC) coupled with radioimmunoassays (RIA).
    • Employed delta sleep-inducing peptide (DSIP) and its radiolabeled form (125I-Tyr-DSIP) as standards.

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  • Quantified peptide carry-over from previous sample applications.

    • Main Results:

    • The shadow phenomenon was observed, indicating peptide carry-over.
    • Carry-over levels reached up to 10% for DSIP.
    • Carry-over was significantly lower (1%) for the radiolabeled 125I-Tyr-DSIP.

    Conclusions:

    • The shadow phenomenon in HPLC-RIA can lead to false positive identification of endogenous peptides.
    • Caution is necessary when interpreting HPLC-RIA results, especially for peptides like DSIP.
    • Methodological adjustments may be needed to mitigate carry-over effects and ensure accurate peptide analysis.