Analysis of O-Phosphoamino Acids in Proteins by Gas Chromatography with Flame Photometric Detection

Abstract

A sensitive and selective method was developed for the analysis of O-phosphoserine (P-Ser), O-phosphothreonine (P-Thr) and O-phosphotyrosine (P-Tyr) in proteins by gas chromatography (GC). After partial acid and base hydrolysis of the protein samples, the liberated O-phosphoamino acids were converted into their N-isobutoxycarbonyl trimethyl ester derivatives and measured by GC with flame photometric detection (FPD-GC), using a DB-1701 capillary column. The calibration lines were linear in the range of 0.1–5 nmol for P-Ser and P-Thr, and in the range of 0.05-1 nmol for P-Tyr, and the detection limits for P-Ser, P-Thr and P-Tyr were about 0.26, 0.18 and 0.30 pmol as injection amounts, respectively. The best hydrolysis conditions for the proteins proved to be 6 M HCl at 110°C for 2 hr to analyze P-Ser and P-Thr, and 5 M KOH at 130°C for 1 hr to analyze P-Tyr. O-Phosphoamino acids in the protein hydrolyzates could be selectively determined by the FPD-GC method without any influence from other amino acids. This method was applied to the analysis of O-phosphoamino acids in several commercial proteins. © 1991, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.