A method for selective isolation of the amino-terminal peptide from alpha-amino-blocked proteins

A method for selective isolation of the amino N-terminal peptide from an alpha-amino (N alpha)-blocked protein is presented. The method consists of four steps. First, epsilon-amino 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. Fourth, uncoupled peptides are purified by reversed-phase chromatography. The amino acid sequence of the peptide including the blocking group can be determined by mass spectrometry. These procedures were successfully tested with three known N alpha-blocked proteins including bovine brain S100 protein, horse cytochrome c, and ovalbumin.