Edman Degradation Sequencing Reaction Conditions
Edman degradation is a method where under weak alkaline conditions, the free α amino acid residue at the N-terminus of a protein peptide chain reacts with phenylisothiocyanate (PITC). The peptide is then treated with acid, allowing only the N-terminal residue to be cleaved as a phenylthiohydantoin (PTH) derivative for sequencing and sequence analysis.
Edman degradation sequencing is completed through iterative cycle reactions, with each cycle containing coupling, cleavage, conversion, and amino acid identification processes, as outlined below:
Coupling conditions: Deliver 12% trimethylamine aqueous solution (pH=8.0-9.0) in gaseous form, deliver n-heptane solution containing phenylisothiocyanate (PITC), and repeat the delivery of alkaline solution. Perform the coupling chemical reaction at 45-48°C, dry with inert gas, and wash with analytical grade solutions of n-heptane and ethyl acetate.
Cleavage conditions: Deliver a few microliters of anhydrous trifluoroacetic acid (TFA) and perform cleavage at 45-48°C, then dry with inert gas to remove acid. Deliver butyl chloride to dissolve the released anilinothiazolinone (ATZ) amino acid, and transport the ATZ amino acid butyl chloride mixture to the converter, repeating the extraction with butyl chloride solvent.
Conversion conditions: Deliver 25%-40% anhydrous trifluoroacetic acid (TFA) water bath solution (conversion medium R4) to the converter, converting at 45-50°C for 10-20 minutes. Dissolve the phenylthiohydantoin amino acid (PTH amino acid) in a suitable solvent mixture, such as 20% acetonitrile aqueous solution with or without sodium acetate additive to adjust pH; this can be directly injected onto the HPLC column of the high-performance liquid chromatography (HPLC) system.
Sequence analysis: Run a blank cycle, run the PTH amino acid and calibrate, compare the amino acid cleaved in each cycle with the PTH amino acid, and compare with amino acids after previous cycles to finally determine the N-terminal amino acid sequence of the protein peptide chain.
Biotech Company uses Shimadzu's Edman sequencing system to provideN-terminal protein sequencing based on Edman degradationservices for researchers and scientific clients. Using our sequencing system, the sequence information of the N-terminal 30 amino acids can be determined. With a specific protein loading system, 60-70 amino acids at the N-terminal can be determined. Biotech Company has also established a platform for N-terminal sequencing using advanced LC-MS/MS technology, which can detect blocked and modified protein termini,complementing N-terminal protein sequencing based on Edman degradationto ensure the smooth progress of N-terminal sequencing services.
Related services:
Protein N/C-terminal sequencing
Biopharmaceutical N/C-terminal sequencing
Full protein sequence determination
Protein sequencing based on the Top-down approach
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