Covalent immobilization of proteins and peptides for solid-phase sequencing using prepacked capillary columns

Abstract

The use of prepacked capillary columns for immobilizing proteins and peptides for solid-phase Edman degradation is described. Capillary tubes with an internal volume of about 30 μl are filled with glass beads bearing isothiocyanato groups (DITC-glass), aminophenyl groups (AP-glass), or aminoethylaminopropyl groups (AEAP-glass) and are sealed with porous plugs. Proteins or peptides in appropriate buffers are introduced into the columns by capillary action and are covalently coupled to the glass beads, either by reaction of lysine side-chain amino groups with DITC-glass, by carbodiimide-mediated reaction of carboxyl groups with AP-glass, or by reaction of homoserine lactone groups with AEAP-glass. Optimization of attachment conditions is described. The capillary columns are loaded into the sequencer and, when sequencing has been completed, are discarded. This technique greatly simplifies polypeptide immobilization and is suitable for microsequencing (<50–1000 pmol) or macrosequencing (1–50 nmol).