The identification of phenylthiohydantoin amino acids by high pressure liquid chromatography

Abstract

A method of analysis of phenylthiohydantoin amino acids was developed using high pressure liquid chromatography. This analysis is useful both quantitatively and qualitatively in protein sequencing, as the residues of the protein degradation are converted to the stable phenylthiohydantoin derivative before identification. When using an automated sequencer to cleave a protein via the Edman degradation, the time of analysis is critical so that the identification is synchronized with the degradation. A gradient elution profile, in which all 20 phenylthiohydantoin amino acids may be identified, has been developed. The analysis requires 25 minutes, which is adequate to allow immediate feedback to the sequencer. Reversed phase chromatography, using a methanol/sodium acetate buffer mixture as the mobile phase was found to be adequate for this purpose, in that it provides a fast, sensitive, and accurate method of identification of phenylthiohydantoins.