High sensitivity amino acid sequence determination. Application to proteins eluted from polyacrylamide gels

Abstract

An automatic solid-phase procedure is described for determining the amino-terminal amino acid sequence of very small quantities of proteins. The sample is covalently attached to an inert support, so that mechanical and physical losses during sequencing are eliminated. High sensitivity is achieved by using an initial coupling with high specific activity phenyl [35S]isothiocyanate followed by a longer reaction with the unlabeled reagent. The radioactive phenylthiohydantoins are identified by autoradiography after 2-dimensional TLC. Unlabeled phenylthiohydantoin-amino acids are added to each fraction to assist in the identification and to act as carriers, hence reducing absorptive and extractive losses of the small quantities of sample. The method may be used on proteins eluted from polyacrylamide gels containing sodium dodecyl sulfate without removal of the detergent. Sequences of up to 20 residues were obtained on quantities of protein [hen egg-white lysozyme, Bacillus stearothermophilus tRNA synthetase and superoxide dismutase and Trypanosoma brucei antigens] ranging from 2.5-70 pmol. Results from proteins of hitherto unknown sequence are included.