Schmidt W E, Conlon J M, Mutt V, Carlquist M, Gallwitz B, Creutzfeldt W
Eur J Biochem. 1987 Feb 2;162(3):467-72. doi: 10.1111/j.1432-1033.1987.tb10663.x.
A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-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 alpha-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 alpha-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 alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.
本文描述了一种用于快速鉴定肽中C末端酰胺化氨基酸的灵敏方法。用内肽酶切割C末端含有α-酰胺基的肽。释放出的片段（寡肽、氨基酸和C末端酰胺化残基）与异硫氰酸苯酯偶联。通过碱性乙酸丁酯从混合物中选择性提取氨基酸α-酰胺的苯硫代甲酰基衍生物，并通过高效液相色谱系统进行鉴定，该系统能够在20 - 50皮摩尔的检测限下快速、完全地分离17种氨基酸酰胺的衍生物。神经激肽 - A（Met - NH₂）、哺乳动物促胰液素（Val - NH₂）、胰多肽（Tyr - NH₂）和肽HI（Ile - NH₂）的C末端α-酰胺在每条肽0.5 - 2纳摩尔的水平上被明确测定。该方法用于表征从猪脑中制备的粗肽级分。通过检测苯硫代甲酰基 - 苯丙氨酰胺在该级分中鉴定出胆囊收缩素 - 58。该方法适用于鉴定纯化肽的C末端α-酰胺化残基，但也可用作一种筛选策略，从复杂的生物提取物中分离出在C末端含有α-酰胺化氨基酸的新型肽。
