N-terminal sequence analysis of polypeptide at the picomole level

Abstract

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins [DABTH] of amino acids were identified by reversed-phase high-pressure liquid chromatography. DABTH are colored compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid DABTH derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins [angiotensin, insulin mellitin, lysozyme, ribosomal protein L24, monoclonal antibody] with < 1 nmol of material.