Analysis of peptides by amino acids composition: contribution of ultraviolet derivative spectrophotometry and retention time prediction

Abstract

Amino acid composition analysis is sometimes used for the identification of peptides obtained from the hydrolysis of a protein of known sequence. Nevertheless, the interpretation of the analysis can be difficult if only one hydrochloric acid hydrolysis can be performed because of the partial destruction of some amino acids and the lack of cleavage of certain peptidic bonds. In this paper we propose combining the analysis of amino acids by two techniques (derivative UV spectrometry and retention time estimation) associated on-line with the purification step of the peptides (reversed-phase HPLC).

A set of 56 peptides from the peptic and chymotryptic hydrolysis of bovine α_s1-casein (α_s1-CN) were analysed in this manner. The difference between the theoretical and observed retention times was 1.9 min, for aromatic amino acids ratios the difference was 4.0%. The ambiguities coming from repeated residues in the sequence or the presence of Trp residues, destroyed by acidic hydrolysis, were solved and a fragment of the α_s1-CN sequence could be attributed to each peptide.