Identification of the C‐terminally α‐amidated amino acid in peptides by high‐performance liquid chromatography

Wolfgang E. SCHMIDT, J. Michael CONLON, Viktor MUTT, Mats CARLQUIST, Baptist GALLWITZ, Werner CREUTZFELDT

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)

Abstract

A sensitive method for the rapid identification of the C‐terminally amidated amino acid in peptides is described. Peptides containing the α‐amide group at the C‐terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C‐terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid α‐amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high‐performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20—50 pmol. The C‐terminal α‐amides of neurokinin‐A (Met‐NH2), mammalian secretin (Val‐NH2), pancreatic polypeptide (Tyr‐NH2) and peptide HI (Ile‐NH2) are unequivocally determined at a level of 0.5—2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin‐58 was identified in this fraction by detection of phenylthiocarbamoyl‐phenylalaninamide. The method is suitable for the identification of the C‐terminal α‐amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an α‐amidated amino acid at the C‐terminus.

Original languageEnglish
Pages (from-to)467-472
Number of pages6
JournalEuropean Journal of Biochemistry
Volume162
Issue number3
DOIs
Publication statusPublished - Feb 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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