IMPROVED GAS-PHASE MICROSEQUENCING USING FLUORESCEIN ISOTHIOCYANATE AND A COVALENT ATTACHMENT OF PEPTIDES ONTO FUNCTIONALIZED MEMBRANES

A fluorescent Edman-type reagent, fluorescein isothiocyanate, was adap ted for gas-phase sequencing. Samples were covalently attached to func tionalized polyvinylidene difluoride (PVDF) membranes or glass filter disks. In the case of sequencing the insulin B chain, the repetitive y ields could be improved up to 98% by a covalent attachment of the pept ide to an arylamine functionalized PVDF membrane from 74% for noncoval ently immobilization on a PVDF membrane. The present method allowed th e identification of 20 amino acid residues from the N-terminus with le ss than 5 pmol of the insulin B chain.