The Edman degradation

As everybody knows, a sequencing machine digests the amino acid chain starting at the N-terminus and it identifies the amino acid derivatives via a connected HPLC. A requirement is a free N-terminus. The Pierce Catalog, for example, describes the chemistry of the Edman degradation of peptides with phenylisothiocyanate. Baumann (1990) compares the effectiveness of different sequencing techniques. 

The most important task of the Edman degradation is still delivering sequence information for the synthesis of oligonucleotides. This preparatory work for protein cloning used to be done as follows. The protein purifier prepared a clean product and handed the preparation to a group specializing in Edman degradation. 

Baumann, M. (1990). Comparative Gas Phase and Pulsed Liquid Phase Sequencing on a Modified Applied Biosystems 477 A Sequencer, Anal. Biochem. 190 198-208. 

Fischer, P. (1992). 25 Jahre Automatisierte Proteinsequenzierung, iVachr. Chem. Techn. Lab. 40 963-971. 

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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... 

A major advance was devised by Pehr Edrnan (University of Lund, Sweden) that has become the standard method for N-terminal residue analysis. The Edman degradation is based on the chemistry shown in Figure 27.12. A peptide reacts with phenyl isothiocyanate to give a phenylthiocarbamoyl (PTC) derivative, as shown in the first step. This PTC derivative is then treated with an acid in an anhydrous medium (Edrnan used nitrornethane saturated with hydrogen chloride) to cleave the amide bond between the N-terminal anino acid and the remainder of the peptide. No other peptide bonds are cleaved in this step as amide bond hydrolysis requires water. When the PTC derivative is treated with acid in an anhydrous medium, the sulfur atom of the C=S unit acts as... 

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. 

Figure 26.4 MECHANISM Mechanism of the Edman degradation for N-terminal analysis of peptides.

PITC (Section 26.6) Phenylisothiocyanate used in the Edman degradation. 

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. 

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. 

The ability to visualize spots on a 2D gel, while useful as a fingerprint, is not the same as protein identification. Protein sequencing by the Edman degradation technique is the classical means of determining... 

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. 

A more useful procedure, in that it allows sequential determination of the A-terminal amino acids in a peptide, is the Edman degradation. This process removes the A-terminal amino acid, but leaves the rest of the chain intact, so allowing further reactions to be applied. The reagent used here is phenyl isothiocyanate. 

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. 

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 length of polypeptide that can be accurately sequenced by the Edman degradation depends on the... 

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. 

Ingenious protein sequenators have been devised to carry out the Edman degradation automatically.242 244-246 Each released phenylthiohydantoin is then identified by HPLC or other techniques. Commercial sequenators have often required 5-20 nmol of peptide but new microsequenators can be used with amounts as low as 5-10 picomoles or less.247 248... 

Because many proteins are modified at the N terminus, blocking application of the Edman degradation, it would be useful to have a similar method for sequencing from the C terminus. It has been difficult to devise a suitable strategy, but there has been some success.252-254... 

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