Edman degradation procedure

Amino acid sequencing may be carried out in a number of ways. The most widely used is the Edman degradation procedure in which phenylisothiocyanate is used to react with the amino acid residue at the amine end of the protein chain. This derivatized residue is removed from the remainder of the protein and converted to a phenylhydantoin derivative which is identified by using, for example, HPLC. 

Enzymes are now being used in conjunction with LC-MS to provide sequence information as an alternative to the Edman degradation procedure. 

Peptide sequencers automatically carry out all the reactions of the Edman degradation procedure under controlled conditions, and a typical scheme is described below. The released N-terminal derivatives are then analysed by reverse-phase HPLC. 

This modification of the DMAA program permitted identification of the first 14 residues. Identification of the polar amino acids in positions 15 and 16 could not be achieved whereas glycine in position 17 could again be recognized. The remaining unknown section of the sequence was elucidated by the manual Edman degradation procedure after tryptic cleavage of C3... 

FIGURE 3-25 Steps in sequencing a polypeptide, (a) Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger s method for identifying the amino-terminal residue is shown here, (b) The Edman degradation procedure reveals... 

To sequence an entire polypeptide, a chemical method devised by Pehr Edman is usually employed. The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact (Fig. 3-25b). The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the amino-terminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluo-roacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The deriva-tized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The use of sequential reactions carried out under first basic and then acidic conditions provides control over... 

The formation of methyl- (MTH) or phenylthiohydantoin (PTH) amino acids is a valuable technique for sequencing of amino acids in peptides and proteins by the Edman degradation procedure [1], HPLC is very useful for the separation of MTH- or PTH-amino acids as adsorption [2,3], reversed-phase [4] and ion-exchange [S] chromatography. 

Figure 3. Modified Edman degradation procedure of globin alkylated by sulfur mustard. (Reprinted from Toxicology and Applied Pharmacology, Vol. 184, D. Noort, H.P. Benschop and R.M. Black, Biomonitoring of Exposure to Chemical Warfare Agents A Review, pages 116-126 (2002), with permission from Elsevier Science.)...

Protein sequence determination uses the standard sequential Edman-degradation procedure to cleave of the N-terminal amino acid of the protein followed by the characterization of the released amino acid in each... 

Lysine may also present problems because its e-amine couples with phenyl-isothiocyanate to form the e-phenylthiocarbamyl derivative, which is sensitive to any trace of peroxides in the sequencer solvents that may decrease its yield. Its chromatographic elution close to leucine frequently requires special attention to gradient conditions as well. Tryptophan, tyrosine, and methionine are sensitive to severe oxidation, although this is usually an infrequent problem, especially with the latter two. Those residues with sidechains containing only carbon and hydrogen (e.g., Ala, Val, lie. Leu, and Phe) are the most stable and are not known to present any complications from the Edman degradation procedure. 

In the past, sequencing of peptides was accomplished using the Edman degradation procedure (see Section 8.2) [3]. Currently, this objective is more often accomplished by interpretation of a peptide s product-ion spectrum. As discussed above, one easy way is to submit the mass spectral data to the database search. Often, this search fails to provide an unequivocal identification of a peptide or protein. Therefore, one needs to resort to a manual interpretation of the mass spectrum. This procedure, known as de novo peptide sequencing, requires a detailed, complete, and... 

FIGURE 23.36 The Edman degradation procedure for sequencing polyamino acids uses phenylisothiocyanate.The amino terminus is identified by the structure of the phenylthiohydantoin formed. Notice that this method does not destroy the peptide chain as the Sanger procedure does. 

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