Dual-detector methods for selective identification of prolyl residues and amide-blocked n-terminal groups in chromatographically separated peptides

Abstract

On-line methods have been developed to screen for peptides containing proline residues or for the presence of an N-terminal blocking group such as an amide. Both methods combine UV detection with post-column reactions, producing a fluorescent product. The fluorescamine post-column reaction is used to detect peptides lacking a free amino terminus. Common blocking groups that can be detected are acetyl, formyl and internally cyclized amino acids such as pyroglutamic. Such groups prevent coupling of phenyl isothiocyanate to the peptide and prevent Edman sequence analysis. The two step, hypochlorite-o-phthalaldehyde post-column reaction detects the presence of proline in the peptide. Proline cannot be readily detected in peptide hydrolyzates by some popular methods of amino acid analysis. These techniques should enhance the success of sequence and composition analysis of collected peptides.