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A gas-liquid solid phase peptide and protein sequenator

R M Hewick, M W Hunkapiller, L E Hood

    The Journal of Biological Chemistry
    |August 10, 1981
    PubMed
    Summary
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    A novel miniaturized protein sequenator utilizes gas-phase reagents for efficient Edman degradation. This instrument provides valuable protein sequence data from minute samples, even for challenging hydrophobic peptides.

    Area of Science:

    • Biochemistry
    • Analytical Chemistry
    • Protein Chemistry

    Background:

    • Traditional protein sequencing methods can be time-consuming and require larger sample amounts.
    • Analyzing hydrophobic peptides presents unique challenges in protein sequencing.

    Purpose of the Study:

    • To develop and characterize a miniaturized protein and peptide sequenator.
    • To improve the efficiency and sensitivity of Edman degradation for protein sequencing.

    Main Methods:

    • Construction of a miniaturized reaction cell for gas-phase Edman degradation.
    • Immobilization of protein/peptide samples using a Polybrene matrix on porous glass fiber discs.
    • Utilizing apolar, liquid-phase solvents to maintain sample integrity during degradation.

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    Main Results:

    • The instrument can yield useful sequence data from as little as 5 pmol of protein.
    • Extended sequence runs (over 30 residues) are possible on subnanomole quantities of SDS-PAGE purified proteins.
    • Successful sequencing of hydrophobic peptides to completion was achieved.
    • High repetitive yield, low reagent consumption, and minimal maintenance were observed.
    • A rapid degradative cycle time of 50 minutes was achieved with a double cleavage program.

    Conclusions:

    • The developed miniaturized sequenator offers a sensitive and efficient platform for protein and peptide sequencing.
    • Its capabilities extend to challenging samples like hydrophobic peptides and those purified by SDS-PAGE.
    • The instrument's design facilitates rapid, low-reagent-consumption, and low-maintenance protein analysis.