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... 

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 ...

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. 

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

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. 

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... 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

Fietzek, P. P., and Kiihn, K. Automation of the Sequence Analysis by Edman Degradation of Proteins and Peptides. 29, 1-28 (1972). 

---