Thiobenzoylation Method of Protein Sequencing: Gas Chromatography/Mass Spectrometric Detection of 5-Acetoxy-2-Phenylthiazoles

Abstract

The thiobenzoylation method of protein sequencing was first proposed in 1967 by Barrett as an alternative to the Edman degradation (Edman, 1950). This method attracted limited interest but facilitated the subsequent development of the closely-related thioacetylation method (Doolittle et al., 1977), wherein the 2-methyl-5(4H)-thiazolone cleavage product is identified by back-hydrolysis to the parent amino acid. The 2-methyl-5(4H)-thiazolone is a volatile analyte which was shown by Previero in 1977 to be potentially suitable for analysis by gas chromatography, either directly or preferably after reaction with an acetylating reagent which affords the corresponding 5-acetoxy-2-methylthiazole. This proposed use of gas chromatography preceded the development of modern capillary gas chromatography columns by several years, however, and consequently column packings which permitted recovery of basic amino acid derivatives were unavailable. We recently reexamined this approach utilizing a capillary gas chromatography/mass spectrometry system and found the method to exhibit considerable potential (Stolowitz et al., 1992). Unfortunately, our efforts to further develop the thioacetylation method have been somewhat hindered by the limited reactivity of thioacetylating reagents and difficulties encountered in the synthesis of a few of the amino acid derivatives for use as analytical standards.