Edman degradation

The Edman degradation is a technology developed by Pehr Edman in 1949 method for protein sequencing.

History

Before the development of proteins with Edman degradation N-terminal labeling with 1-fluoro- 2 ,4-dinitrobenzene ( by Frederick Sanger ) were provided, and then the peptide bonds of the proteins are hydrolyzed, whereby the N-terminal amino acid on the basis of the marking was determined. The dinitrofluorobenzene was later replaced with dansyl chloride, which enabled by fluorescent derivatives have a higher sensitivity of the method. A determination of the amino acid sequence could be achieved only imprecisely to a protein by a time course of amino acid release after addition of an exopeptidase. In the Edman degradation, is in contrast to previous methods, obtained after separation of the derivative of the rest of the protein and can be used in subsequent cycles for the determination of the subsequent amino acids. By use of 4-N, N- Dimethylaminoazobenzen -4' - isothiocyanate ( DABITC ) are colored amino acid derivatives obtained, which facilitates an analysis by thin layer chromatography.

Today, the Edman degradation is no longer widely used. To determine the amino acid sequence of a protein, either the gene sequence of the DNA from a DNA sequencing or from a database of sequenced genomes such as tblast in silico translated into a protein sequence usually. By molecular displays the gene sequence of a protein can be obtained. To identify a protein directly ( partial sequencing ), identifiable parts of a protein can also be studied by mass spectrometry in addition to the Edman degradation. The measured mass of a peptide fragment is compared with all peptide masses, which can be calculated from the genome or exist as Mascot in a database. Along with a simultaneous mass determination of all proteins using MALDI -TOF can be determined as the entire proteome at a given time.

Principle

Edman degradation allowed the determination of the sequence of amino acids ( amino acid sequence) in a peptide by repeated end-group. The peptide chain is thereby progressively reduced. This reaction is used to identify the N-terminal amino acid of a peptide, consisting of the reaction of a peptide with phenyl isothiocyanate, which is also known as a Edman reagent.

Since each amino acid has a different R, maps each a different phenylthiohydantoin derivative (PTH - derivative). The identification of the cleaved as PTH - derivative of the N-terminal amino acid is carried out by chromatography by comparison with a standard. One can successively (each previously shortened by one amino acid at the N -terminal end ) perform other workings on the same protein and determine the amino acid sequence by one. A sequencer is an automated device that allows unattended execution of up to 50 degradation cycles. Since the reaction proceeds with a relative yield of> 98%, on the one part the displaced derivatives of the preceding cycles and on the other hand, the undesired cleavage products from proteins that have been exposed to one or more cleavage cycle, with each cycle, so that after a maximum of 50 cycles of the signal to-noise ratio is illegible.

Mechanism

In the illustrated here, vorgeschlagenene of Zerong Wang, Mechanism, R denotes an alkyl group or hydrogen, Ar denotes an aryl group (usually a phenyl group ) and the wavy line stands for an arbitrarily long continuation Tung of the protein chain:

First, is added to the protein to be analyzed with a 1 Phenylisothiocyanatderivat. This is obtained via an intermediate thioamide 2 About one "bend " it now comes to an intramolecular nucleophilic attack, which a cyclization to Anilinothiazolinon ( ATZ derivative) has the consequence. Thus, the zwitterion 3 is formed from which then a proton transfer the heterocyclic compound 4 is formed. This still carries the remainder of the protein chain, but which is split off in the following step. Is obtained as the product of the remaining protein 5 and the hydantoin derivative 6

This spin-off cycle can now be repeated several times with the residual peptide. Chromatography can now determine the hydantoin derivative 6, and thus the AA sequence. However, only a maximum of 50 cycles are possible. Residues - eg residues of the hydantoin derivative from previous steps - can contaminate the solution so that is no longer traceable, which were split off from them from the current cycle, or which of them come from previous cycles. This sequence determination is no longer to carry out clean. This disadvantage can be circumvented, by decomposing the peptide chain by proteolysis or cyanogen bromide cleavage into several overlapping sub-strings, before starting the sequencing. A cleavage is also necessary for proteins whose N-terminus is modified, for example, N -terminal acetylated proteins. Further disadvantages of the Edman degradation is the high cost, the low sensitivity of approximately one picomolar and a cycle time of about three hours ( with the cyclization rate-determining step).

Modifications

To overcome the disadvantages of loss of sample material during the extraction of the Edman degradation (English wash-out, washing out ' ), it was modified so that you can let him run in the solid phase. This is also called "solid phase support synthesis" or shortly SPSS. In this manner, proteins or short peptides are automatically sequenced. Similarly, immobilized proteins from a Western blot be on a PVDF membrane used, provided only blocking solutions was used without proteins. By using 4 - (1'- Cyanoisoindolyl ) phenylisothiocyanate and phosphorylated amino acids can be detected.

In addition, the Edman degradation has found application in the synthesis of 2- Iminohydantoinen. This reaction is to be exemplified by the reaction of L- leucine and 2- bromophenyl isothiocyanate here. In this synthesis, purity of the product up to 99 % can be achieved.