Covalent attachment of peptides for high sensitivity solid-phase sequence analysis

Abstract

We have developed a method for the high efficiency covalent immobilization of picomole to nanomole quantities of peptides in a form compatible with high sensitivity gas-liquid or solid-phase sequence analysis. Glass fiber filter paper was derivatized with aminophenyltriethoxysilane and peptides were applied to circular disks cut to 1-cm diameters. Peptides were covalently immobilized on the aminophenyl-glass fiber paper through their terminal α-carboxyl groups and amino acid side-chain carboxyl groups by activation with the water-soluble reagent N-ethyl- N′-(3-dimethylaminopropyl)carbodiimide. Disks containing the covalently attached peptide were directly inserted into the cartridge of an automated sequenator for sequence analysis by the Edman degradation. Peptides prepared in this way could be routinely sequenced through to and including the C-terminal amino acid residue, at extraordinarily low backgrounds. The covalent immobilization of peptide fragments allowed far more flexibility in sequencing conditions, including the use of polar extraction solvents to increase the yield of phenylthiohydantoin (PTH)-His and PTH-Arg and the use of alternative Edman-type sequencing reagents with enhanced detectability, such as the chromophoric compound 4-( N,N′-dimethylamino)azobenzene-4′-isothiocyanate. The potential of this high efficiency immobilization method for contributing to the development of sequencing chemistries with enhanced sensitivity is discussed.