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Functionalized membrane supports for covalent protein microsequence analysis.

J M Coull1, D J Pappin, J Mark

  • 1MilliGen/Biosearch, Division of Millipore, Burlington, Massachusetts 01803.

Analytical Biochemistry
|April 1, 1991
PubMed
Summary
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High-yield covalent attachment of peptides and proteins to poly(vinylidene difluoride) membranes enables efficient solid-phase sequence analysis. This method provides reliable protein sequencing with high yields, even for small sample amounts.

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Proteomics

Background:

  • Solid-phase sequence analysis is crucial for protein identification and characterization.
  • Efficient methods for immobilizing peptides and proteins are needed for improved sequencing yields.

Purpose of the Study:

  • To develop high-yield methods for covalently attaching peptides and proteins to poly(vinylidene difluoride) membranes.
  • To enable robust solid-phase sequence analysis of immobilized biomolecules.

Main Methods:

  • Covalent attachment of peptides/proteins to isothiocyanate- or arylamine-derivatized poly(vinylidene difluoride) membranes.
  • Drying solutions of protein/peptide onto membrane disks to facilitate surface reactions.
  • Utilizing N-methylmorpholine for isothiocyanate reactions and water-soluble carbodiimide for arylamine reactions.

Related Experiment Videos

  • Performing Edman degradation on covalently bound polypeptides.

    • Main Results:

    • Achieved initial sequence yields of 33-75% and repetitive yields of 88.5-98.5%.
    • Yields were independent of sample load (20 pmol to >1 nmol).
    • Demonstrated extended N-terminal sequencing (35 residues) and C-terminal sequencing of various peptides and proteins.

    Conclusions:

    • Developed effective covalent attachment strategies for solid-phase protein sequencing.
    • The methods support high-yield, reliable Edman degradation for diverse peptide and protein samples.
    • Applicable to analyzing complex biological samples, including digested proteins.