Fluorescencedetection of aminoacids in the postcleavageconversions for manualsequencing of apeptide

Abstract

A modified Edman degradation method where fluorescent derivatives of aminoacids were generated from the postcleavage products of apeptide is described. In the method, the target peptide was applied onto double glass fiber membranes in a small filter disk (4 mm i.d.) and then treated with small amounts of reagents for the manualsequencing of the peptide. The anilinothiazolinone (ATZ) of N-terminus aminoacid residue after the isolation from the solid-phase membranes was reacted with a primary amine, 4-(1′-cyanoisoindolyl)aniline (CIA), to form a more stable and sensitive fluorescent derivative, phenylthiocarbamoyl–CIA. An average yield of 85% was obtained in neutral pH conditions for the CIA reaction. The ATZ–CIA–aminoacids were separated by reversed-phase liquid chromatography and detected by fluorometry. The lower limits of the detection for aminoacids after the Edman degradation were 0.16 to 0.52 pmol (signal/noise ratio = 3) on the column. The sensitivity was approximately 10 times higher than ultraviolet absorbance detection of phenylthiohydantoin products in the conventional Edman degradation. The suitability of the method was demonstrated by the sensitive manualsequencing of insulin chain B composed of 30 aminoacids.