Isolation and Sequence Determination of Polypeptides at the Picomole Level

Abstract

Increase of sensitivity for protein sequencing depends on the development of micro-methods for amino acid analysis, peptide isolation and sequence determination. While sensitive automatic methods for both amino acid analysis (1,2) and sequence determination (3) are available, there is no straight forward method for peptide isolation. Resolution of a complex peptide mixture usually requires the successive use of combined chromatographic methods (4). The procedure is time-consuming and costly with regard to material since peptide loss occurs with each purification step. Therefore, an ideal peptide isolation method should be not only sensitive but also alone powerful enough to completely resolve peptide mixtures. We have developed new techniques to meet these requirements by precolumn derivatization of amino acids and peptide with chromophoric reagents. The methods used and the results obtained are summarized and discussed in this paper.