What Are the Steps of Edman Degradation?
Edman degradation involves a series of steps to determine the amino acid sequence of a protein or peptide, specifically targeting the N-terminal amino acid. These steps include:
1. Adsorption of the Sample
The protein or peptide to be sequenced is adsorbed onto a solid surface, with glass fiber coated with polybutadiene (a cationic polymer) being a commonly used support material. This is the first of the steps of Edman degradation where the sample is prepared for further chemical reactions.
2. Coupling Reaction
Under weak alkaline conditions (e.g., 12% trimethylamine), phenylisothiocyanate (PITC) reacts with the N-terminal amino acid of the protein or peptide, forming a phenylthiocarbamyl (PTC) derivative. This reaction is conducted in a nitrogen atmosphere and at a temperature of 45-48°C. This is the second step in the steps of Edman degradation, where the PITC specifically reacts with the N-terminal amino group.
3. Cleavage Reaction
In the presence of anhydrous strong acid (e.g., trifluoroacetic acid, TFA), the PTC-peptide undergoes cyclization and cleavage, releasing a thiazolinone phenylthiohydantoin (ATZ) derivative of the N-terminal amino acid. This reaction is carried out at 45-48°C and involves drying under an inert gas. This crucial step is part of the steps of Edman degradation, as it releases the N-terminal amino acid for analysis.
4. Conversion Reaction
The unstable ATZ derivative is converted into a stable phenylthiohydantoin (PTH) derivative in the presence of a dilute acid, typically at 45-50°C for 10-20 minutes. This is the next step in the steps of Edman degradation, where the ATZ is stabilized into a form that can be identified in subsequent analysis.
5. Analysis
High-performance liquid chromatography (HPLC) is used to identify the PTH-amino acid derivatives, and the resulting chromatograms are compared with standard profiles to determine the N-terminal amino acid sequence. This step in the steps of Edman degradation is where the actual sequencing of the amino acid occurs, based on the chromatographic separation of the PTH derivatives.
6. Repetition
This series of reactions is repeated, with each cycle cleaving and identifying a single N-terminal amino acid. The sequence of the N-terminal of the protein or peptide is progressively determined until the full sequence is obtained. This repetition is essential in the steps of Edman degradation, as it allows for the sequential determination of the entire protein sequence.
If the N-terminal amino group of the protein is blocked (e.g., by acetyl, formyl, or pyroglutamic acid), preventing the reaction with PITC, chemical or enzymatic cleavage methods must be employed to generate fragments with free N-terminals. Thus, appropriate preprocessing steps should be carried out for different types of protein samples before proceeding with the steps of Edman degradation.
1. Salt-Free Protein Samples
Edman degradation can be performed directly by applying the sample to a PVDF (polyvinylidene fluoride) membrane. This is a straightforward process in the steps of Edman degradation.
2. High-Salt Protein Samples
Desalting is required prior to analysis. A simple desalting setup can be constructed using detachable filter heads, syringes, PVDF membranes, and EP tubes. After activating the PVDF membrane, the sample, diluted with water, is passed through for desalting, and the membrane can then be analyzed. This step is part of the steps of Edman degradation that ensures the sample is free from interfering salts.
3. Antibody Samples
After separation by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and electroblotting onto a PVDF membrane, the membrane is stained with Coomassie Brilliant Blue. Once air-dried, the heavy and light chain bands are excised for analysis. This step is necessary for the steps of Edman degradation when working with complex protein mixtures like antibodies.
For pyroglutamic acid-cyclized samples, pyroglutamyl aminopeptidase can be used to remove the pyroglutamic acid, followed by SDS-PAGE electrophoresis and electroblotting for further analysis.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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