Selective Isolation of the Amino-Terminal Peptide from α-Amino Blocked Protein

Abstract

The quantitative analyses of both amino (N) and carboxy (C) terminal amino acids are important in estimating the purity or a protein and in confirming the open reading frames of DNA. Edman degradation in a gas-phase (2) sequenator has been the most widely used method for determining the N-terminal sequence of polypeptides. Moreover, structural studies on a variety of eukaryotic proteins, have indicated that as many as 80% of these proteins are α-amino (N α)-blocked (2,3). Proteins having blocked N-termini cannot be sequenced by the Edman degradation. Several methods have been described for removing the modified group or amino acid (4–7), or isolating the N α-blocked peptide (8). However, these methods do not always work efficiently owing to their inherent limitations. A method for selective isolation of the N-terminal peptide from an N α-blocked protein is presented here. Only the N α-blocked peptide lacks an α-amino group in the digests of a target protein when ε-amino (N-ε) is protected prior to digestion, and this serves as the basis for isolation of the blocked peptide (9). The method consists of three steps. First, N-ε groups of lysine residues in the protein are succinylated. Second, the derivatized protein is digested by either enzymatic or chemical cleavage. Third, the digest is subjected to reaction with cyanogen bromide-activated Sepharose. Only the N-terminal blocked peptide fails to react, while the other peptides are covalently bound to the Sepharose.