Improvements to the Automated Sequence Analysis of Short Peptides

Abstract

The Edman degradation (Edman, 1956) for the sequence analysis of peptides and proteins may be divided into three parts. In the first step phenylisothiocyanate is coupled to the N-terminus under alkaline conditions to give a phenylthiocarbamoyl peptide. Secondly, a strong anhydrous acid cyclizes and cleaves off the N-terminal amino acid as its anilinothiazolinone derivative. In the third step the anilinothiazolinone-amino acid is converted into the more stable phenylthiohydantoin derivative and identified. The first two steps were automated by Edman & Begg (1967) in their protein sequenator. The reaction vessel is a spinning glass cup housed in a bell-jar. Reagents and solvents are delivered to the spinning cup and are spread by centrifugal force into a thin film on the inner surface of the cup where the protein had previously been deposited. Excess of added solvents rise over the protein film, reach an annular groove and are scooped out through a Teflon tube. The JEOL sequence analyser used in this work follows this basic principle, but differs from the original Edman & Begg (1967) instrument in certain design features. The JEOL spinning cup is made of Teflon set in stainless steel. Asbefore, reactions and extractions are performed in thin films on the inner surface of the spinning cup. Excess of solvents rise over the protein film to an annular groove from which they are sprayed through a fine hole into a circumferential channel which they leave through a Teflon tube pushed by N2 gas pressure. The spinning cup is housed in a minimum-volume chamber and a space-reducing core projects downwards into the cup. Each solvent and reagent is metered into the cup by its individual pump. Problems with the evaporation of buffers into the bell-jar led Edman & Begg (1967) to introduce the use of the involatile Quadrol [ NNN”’-tetrakis(2-hydroxypropyl)ethylene-