User:Yadi Huo/Edman degradation

Edman degradation is a three-step long-established technique for N-terminal sequencing of proteins, which consists of the coupling of phenylisothiocyanate (PITC) to the α-amino group of a peptide or protein, cleaving the amino-terminal amino acid, and recovering the amino acid for recognition by chromatography. Edman degradation reaction is named after Pehr Edman who first developed and presented it in 1950.

Experimental Reaction Steps

Edman and his group first proposed the procedures for the stepwise degradation of polypeptides in 1950 as following described.

Assumptions and protocols

• The possibility of reaction in which the cleavage of peptide bond occurred parallels the possibility of the formation of hydantoin where the ring is closed.
• The cleavage should not happen at any other places except at the peptide bond adjacent to the carbamyl group in order to achieve the purpose of amino acid sequence recognition.
• The reaction is carried out in an anhydrous, inert solvent to avoid the cleavage caused by hydrolysis. In this case, Nitromethane is used.
• A micromethod is used in which the hydantoin is hydrolyzed to the corresponding amino acid which can be later recognized by the chromatography.

Preparation of the Phenylthiocarbamyl (PTC) peptide

Peptide is prepared in a mixture of pyridine and water solution (1:1) at 40°, and the solution of peptide is added to phenyl isothiocyanate until the solution is saturated. Phenyl isothiocyanate ensures only the N-terminal amino acid is cleaved without damaging the whole peptide, thus, identification of the whole sequence is feasible. Because a high value of pH within limit can increase the reaction rate, so pH value is maintained at 8.6 throughout the reaction by adding an alkali, N sodium hydroxide and a pH indicator, bromothymol blue. Benzene is used repeatedly for extraction to obtain the sodium salt of the PTC-peptide which is then evaporated by a lyophilization procedure. Acetic acid is used to obtain dry PTC-peptide.

Cleavage of the PTC-peptide

Mixture of nitromethane and hydrogen chloride provides an anhydrous environment for the cleavage of the terminal peptide from the PTC-peptide for later recognition. Since the reaction needs to be carried out in an anhydrous solvent, both nitromethane and hydrogen chloride is prepared in advance. Hydrogen chloride is treated with sulfuric acid. Calcium sulfate and phosphorous pentoxide are used respectively to dry nitromethane which is then distillated with dry hydrogen chloride to produce anhydrous solvent of nitromethane-HCL. Dry PTC-peptide from previous step is added to the solvent. The residual peptide cleaved is insoluble nitromethane-HCL thus phenyl thiohydantoin derivative can be extracted by removing the precipitate by filtration.

Identification of the Removed Amino Acid

Amino acid can be recovered by hydrolysis of phenyl thiohydantoin (PHT)-amino acid under acidic condition. Edman utilized alkaline hydrolysis when he and his group published their work in 1950. However, research has shown that the recovery yields of free amino acids are higher when PHT-amino acid is hydrolyzed under acidic condition compared to alkaline hydrolysis. Removed amino acid can be identified by chromatography using pyridine-amyl alcohol as solvent.

Protein Sequenator

Protein sequenator is an instrument presented by Edman and Beggs in 1967 to automate the Edman degradation reaction based on the formation of phenylthiocarbamyl-peptide and triazolinone due to the cleavage of the N-terminal amino acid. Protein sequenator enhanced the efficiencies of amino acid recognition.

Design

A protein sequenator consists of a spinning cup in which the reaction takes place, a feed line through which the reagents and solvents enter from a reservoir that is kept under a constant low pressure by a pressure regulator and a nitrogen supplier, a fraction collector in which the PHT-amino acid is collected one at a time, a waste container, motors and a vacuum pump, and an electronic programming unit that governs all of the functions of the sequenator.

Operation

There are 30 steps in total with some repetitive steps for one complete cycle of each amino acid recognition.

• Reagent stage: Adding corresponding reagent to the reaction cup
• Reaction stage: After adding the reagents, a corresponding reaction takes place
• Vacuum stage: Remove the reaction medium prior to extraction to avoid blockage of the effluent line
• Restricted vacuum stage: The evacuation is only through the capillary bypass
• Delay stage that is between an extraction stage and a vacuum stage: Nitrogen clears the effluent line
• Delay stage that is between vacuum stage and a reagent stage: The pressure is at equilibrium in the bell jar

Comparison with Other Existing Techniques for Protein Sequencing

Edman Degradation	Mass Spectrometry	Sanger Degradtion
Pehr Edman first developed this method in1950	Koichi Tanaka first used mass spectrometry for characterizing ionized protein in 1987	Fredrick Sanger first developed this method in 1945
it involves sequential identification of Amino Acid from N terminals, rest of the peptide chain is left intact for further cycles, so the entire sequence can be identified sequentially.	Protein is ionized in a gas phase prior to its introduction to the mass spectrometry. Characterization of ions is matched against the database	Identification of N-terminal residue of peptide, but the cycle can not be repeated on same peptide chain
Reagent used: Phenyl isothiocynate (PTC)	Reagent used: MALDI or ESI	reagent used: 1-Fluoro-2,4-dinitrobenzene (FDB)
The N terminal amino acid can be identified by reacting the triazolinone derivative with PTC which forms a stable covalent link with free alpha amino group prior to hydrolysis	The peptides matched during protein identification do not necessarily include the N- or C-termini predicted for the matched protein	sanger's reagent react with NH2 group of N-terminus gives a yellow colored derivative on hydrolysis
the end product is identified by high performance liquid chromatography (HPLC)	the end product is identified by mass spectrometry	the end product is identified by gel chromatography
Major limitations: time consuming, harsh conditions, degradation efficiency drops after 50-60 residues	Major limitations: lack of the ability to analyze low-abundance protein samples and to map rare amino acid variants	Major limitations: the entire peptide is damaged during the whole N-terminal

Coupling Analysis and Current Research of Edman Degradation

Even though Edman degradation is gradually replaced by mass spectrometry coupling with HPLC which is now the primary technique for high-throughput protein identification, it still remains as a valuable tool for characterizing N-terminus of proteins.

Swaminathan and his group presented a novel protein sequencing method that Edman degradation can be coupled with selective fluorescence labeling to comprehensively sequence protein samples as complex as mRNA-transcript levels with low abundance and post-translational modifications. By directly visualizing individual peptide or protein that is selectively labeled with a specific identifier fluorophore, millions to billions of amino acids in complex mixtures can be sequenced in parallel.

A computer-aided catalyst named Edmanase for the cleavage step aims to increase the feasibility of Edman degradation by removing the harsh acid catalysis, and to improve compatibility with low adsorption detection surfaces.