Sequencing of Peptides The Edman Degradation

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

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... 

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

Underlined sequences indicate amino acid sequences used for the generation of degenerate primers. Bracketed question marks represent blank cycles from the Edman degradation reaction. Additional sequence was obtained after blank cycles in all cases except the Glu-C-1 and Glu-C-2 peptides. 

Figure 7.5 The Edman degradation method, by which the sequence of a peptide/polypeptide may be elucidated. The peptide is incubated with phenylisothiocyanate, which reacts specifically with the N-terminal amino acid of the peptide. Addition of 6 mol l-1 HCl results in liberation of a phenylthiohydantoin-amino acid derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its constituent amino acid identified by comparison to phenylthiohydantoin derivatives of standard amino acid solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino terminus may be identified. This procedure is repeated until the entire amino acid sequence of the peptide has been established...

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. 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

Recently, Laursen realized the concept of a novel Peptide-Sequencer based on the chemistry of the Edman degradation, adapted to solid phase chemistry. The peptide under investigation is covalently linked to a resin packed into a column. The reactions are carried out by pumping the reagents and solvents through the column as required. This method appears to be particularly suitable for shorter peptides and may be regarded as an excellent supplement to the Sequencer based on Edman s design. 

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... 

Several enzymes catalyze stepwise removal of amino acids from one or the other end of a peptide chain. Carboxypeptidases232 remove amino acids from the carboxyl-terminal end, while aminopeptidases attack the opposite end. Using chromatographic methods, the amino acids released by these enzymes may be examined at various times and some idea of the sequence of amino acids at the chain ends may be obtained. A dipeptidyl aminopeptidase from bovine spleen cuts dipeptides one at a time from the amino terminus of a chain. These can be converted to volatile trimethylsilyl derivatives and identified by mass spectrometry.233 If the chain is shortened by one residue using the Edman degradation (Section 3) and the dipeptidyl aminopeptidase is again used, a different set of dipeptides that overlaps the first will be obtained and a sequence can be deduced. Carboxypeptidase Y can be used with MALDI mass spectrometry to deduce the C-terminal amino acid sequence for a peptide. However, He and Leu cannot be distinquished. 

GLC is an important adjunct to protein sequence determination. Automatic "sequenators" based upon the approach developed by Edman are available and have been described in detail by Niall (60). The Edman degradation, summarized in Equation 9.5, makes use of methyl or phenylisothiocyanate which reacts with the N-terminus of a peptide. Exposure of the isothiocyanate derivative of the protein to acid results in cleavage of the terminal amino acid as a thiaxolinones and exposure of the next amine group on the peptide. Thus, the process can be repetitively carried out, each amino acid removed from the peptide, in a sequential manner. Thiazolinones rearrange in acid medium to form thiohydantoin derivatives of amino acids, some of which may be directly gas chromatographed others must be derivatized typically as trimethylsilyl derivatives. 

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