Edman breakdown

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The Edman degradation is one of the Swedish biochemist Pehr Edman developed in 1949 method for protein sequencing . The sequencing takes place - in contrast to the Schlack-Kumpf breakdown - starting from the N -terminal end of the protein.

history

Before the development of the Edman degradation, proteins were provided with 1-fluoro-2,4-dinitrobenzene (according to Frederick Sanger ) by N- terminal labeling , then the peptide bonds of the proteins were hydrolyzed, whereby the N- terminal amino acid was determined on the basis of the labeling . The dinitrofluorobenzene was later replaced by dansyl chloride , which, thanks to fluorescent derivatives, made the method more sensitive . A determination of the amino acid sequence could only be achieved imprecisely by the time course of the amino acid release after adding an exopeptidase to a protein. In the case of Edman degradation, in contrast to the previous methods, the rest of the protein is retained after the derivative has been split off and can be used in subsequent cycles to determine the subsequent amino acids.

Edman mining is no longer widely used these days. In order 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 is translated into a protein sequence. The gene sequence of a protein can be obtained through molecular displays . In order to identify a protein directly ( sequencing ), identifiable parts of a protein can be examined not only by Edman degradation but also by mass spectrometry . The measured mass of a peptide fragment is compared with all peptide masses that can be calculated from the genome or appear in a database such as Mascot . Together with a simultaneous mass determination of all proteins using MALDI-TOF , the entire proteome can be determined at a specific point in time.

principle

Edman degradation allows the order of amino acids (the amino acid sequence) in a peptide to be determined by repeated end grouping. The peptide chain is gradually broken down. This reaction is used to identify the N -terminal amino acid of a peptide and consists of the reaction of a peptide with phenyl isothiocyanate , which is also known as Edman's reagent .

During Edman degradation, the N -terminal amino acid of the protein (left) is split off under the action of phenyl isothiocyanate to form the protein shortened by one amino acid and a phenylthiohydantoin derivative (PTH derivative). R = alkyl radical or hydrogen , Ar = phenyl radical , serpentine line = continuation of the protein chain of any length.

Since each amino acid has a different residue R , each forms a different phenylthiohydantoin derivative (PTH derivative). The N- terminal amino acid split off as PTH derivative is identified by chromatography by comparison with a standard. You can successively carry out further degradations on one and the same protein (previously shortened by an amino acid at the N -terminal end) and thus gradually determine the amino acid sequence. A sequenator is an automated device that allows the unattended execution of up to 50 degradation cycles. Since the reaction proceeds with a relative yield of> 98%, on the one hand the entrained derivatives from previous cycles and on the other hand the undesired cleavage products of proteins that have undergone one or more cleavage cycles increase with each cycle, so that after a maximum of 50 cycles the signal -Noise ratio becomes illegible. A cycle takes one to three hours, depending on the variant.

mechanism

In the mechanism proposed by Zerong Wang shown here, R denotes an alkyl radical or hydrogen, Ar denotes an aryl radical (usually a phenyl radical ) and the serpentine line stands for an arbitrarily long continuation of the protein chain:

Edmann dismantling (mechanism)

First, the protein 1 to be analyzed is mixed with a phenyl isothiocyanate derivative. This gives thioamide 2 via an intermediate stage . A “bend” now leads to an intramolecular , nucleophilic attack, which results in cyclization to anilinothiazolinone (ATZ derivative). This forms the zwitterion 3 from which the heterocyclic compound 4 is then formed via proton transfer . This still carries the remaining protein chain, which, however, is split off in the following step. The product obtained is the remaining protein 5 and the hydantoin derivative 6 .

This cleavage cycle can now be repeated several times with the residual peptide. The hydantoin derivative 6 and thus the AA sequence can now be determined by means of chromatography . However, only a maximum of 50 cycles are possible. Residues - e.g. B. residues of the hydantoin derivative from previous steps - can contaminate the solution so that it can no longer be traced which of them were split off from the current cycle or which of them came from previous cycles. As a result, a sequence determination can no longer be carried out properly. This disadvantage can be avoided by splitting the peptide chain into several overlapping partial chains by proteolysis or cyanogen bromide before starting the sequencing. Cleavage is also necessary in the case of proteins whose N terminus is modified, e.g. B. N -terminal acetylated proteins or proteins with N -terminal pyroglutamic acid . Further disadvantages of Edman degradation are the high costs, the low sensitivity of around one picomole and a cycle time of around three hours (with the cyclization as the rate-determining step).

Modifications

In order to avoid the disadvantages of a loss of sample material during the extractions of the Edman degradation (English wash-out ), it was modified so that it can run off in the solid phase. One also speaks of “ solid-phase support synthesis ” or SPSS for short. In this way, short proteins or peptides are automatically sequenced. Proteins immobilized on a PVDF membrane from a Western blot are also used, provided that only blocking solutions without proteins were used. By using 4- N , N- dimethylaminoazobenzene-4'-isothiocyanate ( DABITC ), colored amino acid derivatives can be obtained, which facilitates analysis by thin layer chromatography . By using 4- (1'-cyanoisoindolyl) phenylisothiocyanate, phosphorylated amino acids can also be detected.

In addition, the Edman degradation has found application in the synthesis of 2-iminohydantoins. This reaction is to be shown here as an example with the reaction of L - leucine amide and 2-bromophenyl isothiocyanate. With this synthesis, purities of the product of up to 99% can be achieved.

Example Edman degradation

Individual evidence

  1. ↑ P. Edman: A method for the determination of amino acid sequence in peptides . In: Arch. Biochem. tape 22 , no. 2 , 1949, p. 475 , PMID 18134557 .
  2. ↑ Paula Yurkanis Bruice: Organic Chemistry . 5th Edition, Pearson Education Inc., 2007, ISBN 978-3-8273-7190-4 , pp. 1200-1201.
  3. ↑ a b c Z. Wang (Ed.): Comprehensive Organic Name Reactions and Reagents. 3 volume set. John Wiley & Sons, Hoboken, NJ, 2009, ISBN 978-0-471-70450-8 , pp. 954-955.
  4. ^ A b Gavin E. Reid, Shane E. Tichy, James Pérez, Richard AJ O'Hair, Richard J. Simpson: N-Terminal Derivatization and Fragmentation of Neutral Peptides via Ion — Molecule Reactions with Acylium Ions: Toward Gas-Phase Edman Degradation? In: J. Am. Chem. Soc. No. 123 , 2001, p. 1184-1192 , doi : 10.1021 / ja003070e .
  5. ^ Richard A. Laursen: A Solid-State Edman Degradation . In: J. Am. Chem. Soc. No. 88 , 1966, pp. 5344-5346 , doi : 10.1021 / ja00974a069 .
  6. ↑ JY Chang, EH Creaser: A novel manual method for protein-sequence analysis. In: Biochem J. 157, No. 1, 1976, pp. 77-85, PMID 822842 , PMC 1163818 (free full text).
  7. ↑ Takayuki Shibata, Moses N. Wainaina, Takayuki Miyoshi, Tsutomu Kabashima, Masaaki Kai: A manual sequence method of peptides and phosphopeptides using 4- (1'-cyanoisoindolyl) phenylisothiocyanate. In: Journal of Chromatography A. 1218, No. 24, 2011, pp. 3757-3762, doi : 10.1016 / j.chroma.2011.04.040 , PMID 21531425 .
  8. ↑ Ghotas Evindar, Robert A. Batey: Peptide Heterocycle Conjugates: A Diverted Edman Degradation Protocol for the Synthesis of N-Terminal 2-Iminohydantoins . In: Org. Lett . tape 5 , no. 8 , 2003, p. 1201-1204 , doi : 10.1021 / ol034032d .