Improved Gas-Phase Microsequencing Using Fluorescein Isothiocyanate and a Covalent Attachment of Peptides onto Functionalized Membranes

Abstract

A fluorescent Edman-type reagent, fluorescein isothiocyanate, was adapted for gas-phase sequencing. Samples were covalently attached to functionalized polyvinylidene difluoride (PVDF) membranes or glass filter disks. In the case of sequencing the insulin B chain, the repetitive yields could be improved up to 98% by a covalent attachment of the peptide to an arylamine functionalized PVDF membrane from 74% for noncovalently immobilization on a PVDF membrane. The present method allowed the identification of 20 amino acid residues from the N-terminus with less than 5pmol of the insulin B chain.