NTerminalsequenzierung

NTerminalsequenzierung, also known as Edman degradation, is a biochemical technique used to determine the amino acid sequence of a protein or peptide from its N-terminus. Developed by Frederick Sanger in the 1940s and later refined by Pehr Edman, this method involves a cyclic process of chemical reactions. In the first step, the N-terminal amino acid is selectively reacted with phenylisothiocyanate (PITC) under alkaline conditions, forming a phenylthiocarbamyl (PTC) derivative. This derivative is then cleaved from the rest of the peptide chain under acidic conditions, yielding an anilinothiazolinone (ATZ) derivative of the N-terminal amino acid and a shortened peptide. The ATZ derivative is subsequently converted to a more stable phenylthiohydantoin (PTH) amino acid, which can be identified using chromatography, typically high-performance liquid chromatography (HPLC). The shortened peptide is then subjected to the same cycle of reactions, allowing for the sequential identification of amino acids from the N-terminus. This process can be automated, making it a relatively efficient method for sequencing short to medium-length peptides. However, its effectiveness is limited by the accumulation of side reactions and degradation of the peptide with each cycle, making it less suitable for very long proteins. NTerminalsequenzierung played a crucial role in early protein structure determination and is still used today, particularly for verifying protein identity and for sequencing peptides that have been produced by recombinant DNA technology.