Edman technique

The AT-terminal amino acid can be ascertained by the Sanger method. Enzymic cleavage using either chymotrypsin or trypsin will break the peptide into smaller fragments. The fragments obtained will be different, depending on the enzyme and its specificity. The smaller fragments are then each sequenced by the Edman technique. C-Terminal residues in the smaller... 

These techniques have been used successfully in the micro-Zdman degradation of the enzyme mouse sarcoma dihydrofolate reductase to obtain the amino acid sequence of the first 25 amino acids 455). Similarly, RPC has been used in coqjunction with the automated Edman technique for sequencing 32 residues of myoglobin 456). Methionine and its oxidation products, methionine sulfoxide and methionine sulfone, in methionine fortified foods have been analyzed as their dansyl derivatives 457). Lysine has been determined as its dansyl derivative in a study in which the stability of lysine in fortified wheat flour was evaluated (458). 

Fatty acylation of proteins is a common posttranslational modification that blocks sequencing of the N-terminal amino acids by Edman techniques. Mass spectrometry can play an essential role in such sequencing, but it is not always trivial to locate the blocked N-terminal peptide. Besides acetylation, acylation of amino acids with longer-chain fatty acids (e.g., myristic acid) has been reported. 

Sequential analysis can be accomplished by using the Edman technique. Treatment of an intact polypeptide with phenylisothiocyanate derivatizes the N- amino acid leaving the rest of the peptide intact for further Edman degradation. Large chains must be fragmented into shorter peptides, more easy to work with chemically. Cleavage of peptide bonds at specific amino acid residues is accomplished using enzymes such as trypsin (Lys, Arg), chymotrypsin (aromatics), and carboxypeptidase (C-terminus amino acids). 

The Edman degradation, a process by which amino acids are removed stepwise from the NH 2-terminus of peptides and proteins, has evolved since its introduction (Edman, 1950) to the point where it is currently the most valuable tool for protein sequence analysis. Several innovations have led to its widespread use. One of the first was the dansyl Edman technique (Gray, 1967), which is useful for sequencing at the nanomole level. Accelerated manual procedures have been developed by Niall and Potts (1970) and Tarr (1975). During the past decade the rate of sequencing has been... 

DNS-Edman refers to the danysl Edman technique and sub-Edman to the subtractive Edman approach to peptide sequencing. Non-automated Edman degradation of peptides coupled with PTH identification is referred to as manual Edman. L/P and S/P refer to liquid pha.se and solid phase automatic sequencing, respectively. 

Improved methods for end group analysis have become available, notably the use of dansyl chloride (5-dimethylaminonaphthalene-l-sulphonyl chloride) for labelling iV-terminal amino acids and the Edman technique. Dansyl-amino acids fluoresce strongly in ultraviolet light and can be detected in very small amounts so that the method is much more sensitive than Sanger s original DNP technique. 

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). 

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... 

N-terminal sequencing is normally undertaken by Edman degradation (Figure 7.5). Although this technique was developed in the 1950s, advances in analytical methodologies now facilitate fast and automated determination of up to the first 100 amino acids from the N-terminus of most proteins, and usually requires a sample size of less than 1 umol to do so (Figure 7.6). 

Analogous techniques facilitating sequencing from a polypeptide s C-terminus remain to be satisfactorily developed. The enzyme carboxypeptidase C sequentially removes amino acids from the C-terminus, but often only removes the first few such amino acids. Furthermore, the rate at which it hydrolyses bonds can vary, depending on what amino acids have contributed to bond formation. Chemical approaches based on principles similar to the Edman procedure have been attempted. However, poor yields of derivatized product and the occurrence of side reactions have prevented widespread acceptance of this method. 

Edman Degradation. This technique requires more material than MS-based sequencing and its sensitivity decreases with the number of amino acids detected. The use of Edman degradation sometimes allows determination of those N-terminal amino acids that were not detected during MS sequencing. 

Figure 10.15 Solid-phase peptide sequencing procedure based on Edman degradation technique.

Sequencing peptides with tandem mass spectrometry was carried out in the early 1980s (Biemann, 1986 Hunt et al., 1986 Hall et al., 1993). Usually the sensitivity and the lengths of sequences achievable were not sufficient to compete with Edman sequencing techniques. In 1988 and 1989, two efficient cold ionization techniques for large molecules were discovered MALDI (Karas and Hillenkamp, 1988) and the electrospray... 

Applicability of CZE to the Edman phenylthiohydantion derivatives of amino acids (140) is limited because the neutral amino acids cannot be resolved by this method and by the reduced thickness of the sample requiring relatively high concentrations of the fluorescent material for detection. These limitations may be overcome by a micellar technique that confers mobility to neutral 140 species and by application of thermotropic detection that allows one to detect a few tens of fmol of the derivative, obtained after injecting ca 0.5 nL, at a concentration of ca 1 p-M330. 

Gerhart M.J., Balland A., and Paxton RJ. (1997), Evaluation of CNBr digestion followed by Edman sequencing as a tool for assessing methionine susceptibility to oxidation in native molecules, ABRF 97 Techniques at the Genome-Proteome Interface, Baltimore, MD. 

N-terminal sequencing is normally undertaken by Edman degradation (Figure 3.35). Although this technique was developed in the 1950s, advances in analytical methodologies now facilitate... 

Sequencing by both Edman sequence analysis and mass spectrometric analysis can be complimentary and where one may fail in yielding information concerning the synthesis problem, the other may succeed. This is because every peptide has different properties and each technique has advantages and disadvantages. However, sequence analysis is expensive and time consuming, which must be taken into consideration when solving the problems of peptide synthesis, especially those of routine peptide synthesis. 

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... 

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