The Edman Degradation and Automated Sequencing of Peptides

The Edman Degradation and Automated Sequencing of Peptides 1144 Peptide Mapping and MALDI Mass Spectrometry 1146 25.24... 

Modem methods of peptide sequencing follow a strategy similar to that used to sequence insulin but are automated and can be carried out on a small scale A key feature is repetitive N terminal identification using the Edman degradation... 

Fig. 1 Summary of the data used to establish the complete amino acid sequence of Er-1 mating pheromone. The peptides have been designated and numbered according to the type of digest ana the theoretical order in which they appear in the sequence. Designations are CNBr, cyanogen bromide T, trypsin V8, . aureus V8 protease CT, chymotrypsin. Peptiaes indicated by two numbers connected with a hyphen result from partial cleavage. Residues directly identified by automated Edman degradation and carboxypeptidase Y digestion (CP-Y) are marked by right and left arrows, respectively, residues identified by amino acid composition are indicated by dashed lines. Taken from ref. 13 and reproduced by permission of the American Society of ...

The structure and properties of peptides and proteins depend critically upon the sequence of amino acids in the peptide chain. The first complete amino acid sequence of a protein, that of insulin (51 amino acid residues), was determined by F. Sanger in 1953. The process is now performed using automated protein sequencers, and involves step-by-step identification of amino acids at the N terminus of the protein using a chemical process known as Edman degradation. 

Because C-terminal amides have often been reported in insect neuropeptides (8), a 33 residue C-terminal amide consistent with the sequence data was synthesized by solid-phase methods. The peptide was purified by HPLC and its structure confirmed by automated Edman degradation. Californium-252 time-of-flight plasma desorption mass spectrometry provided additional evidence for the structure via a very broad peak for the singly-charged molecular ion at m/z 3902-3906. Because the calculated MW of Hez-PBAN (3899.6, based on the most abundant ion in the isotope cluster) was seen to differ from that observed in the mass spectrum of the isolated native peptide by ca. 32, it was presumed that the native peptide had undergone oxidation of both its methionines to their respective sulfoxides during the course of its isolation and purification. 

H- and 13C-NMR data have been reported for diagnostic purposes in direct analysis of phenylthiohydantoin amino acid derivatives (PTH) produced in the Edman degradation of peptides and proteins.189-193 The insensitivity of 3H-NMR spectroscopy constitutes a major hurdle for its application in the sequence study of peptides.194,195 Alternatively, identification of the cleaved amino acids in the automated Edman degradation has been solved in some cases by using IR,196-198 mass,199 and gas chromatographic techniques.200... 

Describe the sequential Edman degradation method and the automated determination of the amino acid sequences of peptides. 

Edman, Pehr Victor, 1916-1977 (pp. 118,240, Plate 15) born in Stockholm in 1916, matriculation examination in 1935, studied medicine at the Karolinska Institute Medical School in Stockholm from 1935, Bachelor of Medicine in 1938, graduation as a physician in 1946. Concurrently with his studies in medicine he started his training in biochemistry with Erik Jorpes, for a short time also with Hugo Theorell, and soon started a project on angiotensin that led to a MD thesis. Then he widened his experience in protein chemistry during one year at the Rockefeller Institute in Princeton with Northrop and Kunitz (crystallization of proteolytic enzymes). On his return to Sweden, Edman was awarded an associate professorship in Lund in 1947 where he conducted his stepwise peptide degradation work (p. 118) between 1950 and 1956. In 1957 Pehr Edman accepted an offer to be Director of Research at St. Vincent s School of Medical Research in Melbourne, Australia, where he remained for 15 years, during which the work on an automated sequence analyzer was finished in 1967. From 1972 until his death from a brain tumor in 1977 he was Director of the Department of Protein Chemistry of the Max-Planck-Institute for Biochemistry in Martinsried near Munich. 

Traditionally, proteins were initially characterized by de-novo sequencing using automated Edman degradation and amino acid composition analysis. Today, however, these techniques tend to be replaced by MS, which not only provides more flexibility and sensitivity but is also amenable to the analysis of protein and peptide mixtures. Tandem mass spectrometry (MS/MS) is used for amino acid sequencing of peptides. MALDI-MS/MS is very powerful for peptide characterization and identiflcation via sequencing and sequence database searching. 

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