Gas-phase Microsequencing of Peptides and Proteins with a Fluorescent Edman-type Reagent, Fluorescein Isothiocyanate

Abstract

A fluorescent Edman-type reagent, fluorescein isothiocyanate (FITC), was adapted to a gas-phase sequencer fitted with a miniature reaction chamber. Fluorescein thiohydantoin amino acids were identified by on-line gradient HPLC with fluorescence monitoring. The optimized protocol for the coupling reaction using FITC and phenylisothiocyanate, and the degree of washing to remove the excess reagents were established. Myoglobin (5 pmol) and lysozyme (5 pmol) were analyzed by the sequencer to obtain a 35-51% initial yield (I. Y. ) and 82-88% repetitive yield (R. Y. ) for the former, and 51-66% I. Y. and 91-92% R. Y. for the latter. These figures permitted 20-25 amino acid residues to be identified from the N-terminus of 5 pmol samples applied to the sequencer.