Edman method

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

Substitution of alkaline cyanates by isocyanates allows the preparation of 3-substituted hydantoias, both from amino acids (64) and amino nitriles (65). The related reaction between a-amino acids and phenyl isothiocyanate to yield 5-substituted 3-phenyl-2-thiohydantoiQS has been used for the analytical characterization of amino acids, and is the basis of the Edman method for the sequential degradation of peptides with concomitant identification of the /V-terminal amino acid. 

While the first 20-30 residues of a peptide can readily be determined by the Edman method, most polypeptides contain several hundred amino acids. Consequently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein s a-amino group blocked , or unreactive with the Edman reagent. 

Probtom 21.30 Describe the Merrifield solid-phase synthesis when carried out in the reverse order of the classical Sanger and Edman methods. <... 

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

The Edman method of NH2-terminal amino acid analysis. 

B 2. The new reagent 4-W,W-dimethylaminoazobenzene-4 -isothiocyanate (DABITC) is often used for the Edman method of protein sequence analysis. Write a reaction showing this use of the reagent. 

Today s sequence techniques have achieved 9-10 orders of magnitude higher detection limit for PTH-amino acid than Edman s first report, in which he described the sequence of Ala-Leu-Gly with 100 pmol. Matsuo et al. succeeded in determing the structure of FSH-RH (LH-RH), a decapeptide hormone, in 1971 by using the Dansyl Edman method.83 This historical work used 200 nmol of FSH-RH purified from 165,000 porcine hypothalami and led a principal investigator, A. Schally, to win the Nobel Prize. Today, we would only need less than one hundred porcine hypothalami to complete the same job on a state-of-art sequencer. Those who plan the structural analysis of even barely detectable amounts of a peptide or protein are urged to start the plan such study could open up a new area in biochemistry. 

After the isolation of the peptides from the peptide map, the structure of individually isolated peptides needs to be determined. The building blocks of a peptide are amino acids and the order of occurrence in a peptide is done by a process called sequencing. Sequencing is the ability to cleave only one amino acid at a time from the peptide backbone. Total amino-acid sequencing can be accomplished by the Edman method, followed by identification of each cleaved amino-acid group by HPLC. After LC purification... 

Protein chemists have sought a practical, automated carboxy-terminal (C-termi-nal) sequencing method for many years. In the biophannaceutical industry, C-ter-minal sequence information can be essential for the early characterization of recombinant proteins, as well as for routine batch analysis. Several automated chemical degradation schemes, analogous in principle to the Edman method for amino-terminal (N-terminal) sequencing, have been proposed (1,2). In 1992, we introduced a novel alkylation method for C-terminal protein sequencing (3,4). 

This was first foimd by Sanger et al. (1955) in a peptide from insulin and was observed with other peptides by Hirs et al. (1956) and Smyth et al. (1962). The reaction appears to occur when acidic buffers or dilute acids are employed for isolation of peptides. Conversion of the cyclic pyrrolidone carboxyl residue to a glutamyl residue is obtained on mild hydrolysis in dilute acids or alkalies. The cyclization reaction leads to difficulties when sequence methods are used which proceed from the amino-terminal end of a peptide. In addition, this reaction can occur when an internal glutamine residue becomes amino-terminal in the course of stepwise sequence analysis under acidic conditions, as in the Edman methods. An incorrect sequence for a peptide from ribonuclease was deduced as the result of cyclization of amino-terminal glutamine and acidic destruction of serine and threonine in the same peptide (Smyth et al., 1962). 

In principle, it should be possible to sequence an entire protein by using the Edman method. In practice, the peptides cannot be much longer than about 50 residues. This is so because the reactions of the Edman method, especially the release step, are not 100% efficient, and so not all peptides in the reaction mixture release the amino acid derivative at each step. For instance, if the efficiency of release for each round were 98%, the proportion of "correct" amino acid released after 60 rounds would be (0.98 0), or 0.3—a hopelessly impure mix. This obstacle can be circumvented by cleaving the original protein at specific amino acids into smaller peptides that can be sequenced. In essence, the strategy is to divide and conquer. 

The peptides obtained by specific chemical or enzymatic cleavage are separated by some type of chromatography. The sequence of each purified peptide is then determined by the Edman method. At this point, the amino acid sequences of segments of the protein are known, but the order of these segments is not yet defined. How can we order the peptides to obtain the primary structure of the original protein The necessary additional information is obtained from overlap... 

P. Edman, Method for determination of the amino add sequence in peptides, Acta Chem.Scand. 4 283 (1950). 

Obviously, incomplete reaction and losses during manipulation prevent the yield of 3-phenyl-2-thiohydantoin from reaching 100%. With each cycle of the Edman method, the yield of product derived from the newly exposed TV-terminus will decrease. In addition, small amounts of the 3-phenyl-2-thiohydantoins corresponding to earlier positions in the sequence will be formed as a consequence of incomplete reaction at each cycle. If the fraction of peptide that reacts with phenyl isothiocyanate and gives the relevant 3-phenyl-2-thiohydantoin is x, then the yield at any stage is... 

Although the Edman method is by far the most common method of sequencing peptides from the /V-terminus, some enzymic methods are used occasionally. Several aminopeptidases are available commercially, which differ in their specificities. One aminopeptidase from porcine kidney preferentially releases amino acids such as leucine with hydrophobic side-chains. This enzyme does not release /V-terminal Arg or Lys or any amino acid that is followed by Pro. Another enzyme, aminopeptidase M, which is obtained from the microsomal fraction of porcine kidney cells, is less specific and perhaps more useful. It is advisable to examine aliquots of the hydrolysate at intervals by chromatography to determine the order in which amino acids are being released. 

In order to obtain fragments from a protein of a suitable size for sequencing by the Edman method, it is desirable to effect specific cleavage adjacent to the rarer amino acids. Numerous chemical methods of specifically cleaving proteins have been devised, but only a few are in common use. 

Tandem mass spectrometry is replacing the Edman method as a sequencing tool. Due to the fact that peptides possess both basic and acid functional groups, they readily form [M + H]+ and [M - H] ions under FAB, ESI, and MALDI conditions, which can be subjected to CID to yield the partial or complete sequence of the peptide. 

Schroeder, W., Degradation of peptides by the Edman method with direct identification of the PTH-amino acid, Meth. Enzymol., 11, 445-475, 1967. 

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