Edman Degradation Sequencing: A Reliable N-terminal Protein Analysis Method

Introduction: Precise Identification of N-terminal Sequences Lays the Foundation for Protein Research

In protein research,the precise identification of amino acid sequences is the first step in understanding their function and structure. Although modern mass spectrometry has excelled in the field of proteomics, a classic sequence analysis method remains indispensable in certain specific scenarios—Edman degradation. This chemical method for sequentially analyzing protein sequences from the N-terminus is still widely used due to its high specificity and accuracyin key areas such as protein expression verification, N-terminal modification detection, and quality control. As an important part of protein sequencing services,Edman degradation provides researchers with a low-bias, high-fidelity method for N-terminal sequencing. At BTP Biotech, we combine automated Edman sequencing platforms with high-quality purification processes to offer precise and reliable N-terminal sequence analysis solutions, supporting basic research and biopharmaceutical development.

 

1. What is Edman Degradation?

Edman degradation is a sequential N-terminal sequencing method based on chemical reactions, proposed by Pehr Edman in 1950. Its core principle involves using phenyl isothiocyanate (PITC) to react with the free amino group at the N-terminus of proteins to form a cleavable derivative, which is then released and identified as the first amino acid residue through acid hydrolysis. This process can be repeated while maintaining the integrity of the remaining peptide chain, thus decoding the N-terminal sequence of proteins step by step.

Three core steps of the Edman reaction:

1. PITC Labeling: Under alkaline conditions, PITC combines with the N-terminal amino group of the peptide chain to form phenylthiocarbamyl.

2、Acid Hydrolysis: Anhydrous acid is added to catalyze the cleavage, releasing the N-terminal amino acid in a recognizable form (PTH-AA).

3、Sequence Cycling: The remaining peptide chain re-enters the next cycle, repeating the above steps.

Currently, variousautomated Edman sequencershave been developed in the market, allowing forsequential analysis of up to 30 amino acid residueswith good reproducibility and stability.

 

2. Advantages and Limitations of Edman Degradation

Although the rise of mass spectrometry has opened new horizons for proteomics, Edman degradation still retains its place in several key scenarios due to itscertainty of chemical reactions and accuracy of sequence sites.

1. Advantages:

(1) High specificity: Reacts only with the free amino group at the N-terminus, avoiding interference from other sites.

（2）Sequence Position Determination: Each cycle starts from the N-terminus, ensuring accurate sequence positioning.

（3）Suitable for Protein Expression Verification: Can be compared with theoretical sequences to confirm the correctness of expression products.

（4）Detection of Post-Translational Modifications: Can directly determine whether the N-terminus has acetylation, formylation, or other blocking modifications.

 

2、Limitations:

(1) High Sample Requirements: Must be purified protein, and the N-terminus cannot be blocked or modified.

（2）Limited Sequencing Length: Typically able to sequence up to 20~30 amino acids.

（3）Low Throughput: Not suitable for complex mixtures or high-throughput analysis.

Therefore, during the experimental design phase, researchers need to weigh the applicability of different sequencing technologies to obtain the most informative data.

 

3. Applications of Edman Sequencing in Modern Protein Research

1. Biopharmaceutical Development and Quality Control

In the production of recombinant protein drugs,the N-terminal sequence is an important indicator of drug consistency, stability, and correct expression.Edman sequencing can be used for:

• verifying if the N-terminal amino acid of the expressed product matches the designed sequence

• detecting whether the signal peptide has been successfully cleaved

• monitoring the consistency of expression across different batches

 

2. Verification of translation initiation sites

When constructing expression vectors, researchers often want to confirm whether translation starts from the expected start codon. Edman degradation, by directly reading the N-terminal sequence, can accurately verify any shift in the translation initiation site.

 

3. Analysis of protein N-terminal modifications

Some proteins may undergo N-terminal acetylation, formylation, carbamylation, and other modifications.When Edman degradation cannot proceed, it indicates the presence of blocking modifications, suggesting the need for further structural confirmation using mass spectrometryto construct a complete N-terminal modification profile.

 

IV. Edman Sequencing and Mass Spectrometry: Complementary, Not Substitutive

In modern proteomics,Edman sequencing and mass spectrometry are not in competition but are complementary. Mass spectrometry offers high throughput and broad coverage, allowing rapid scanning of complex samples; whereas Edman degradation providesprecision, low background, and clear sequence positioningcharacteristics that provide precise confirmation for key proteins.

Common strategies include:

1. Mass spectrometry first, followed by Edman: After initial protein screening, Edman verification is conducted on key bands

2、EEdman to screen modified proteins, then LC-MS/MS to locate modification sites

3. Using mass spectrometry and Edman simultaneously in antibody validationto ensure N-terminal consistency

 

The advantages of N-terminal sequencing services by BioPark Biotech

At BioPark Biotech, we understand the importance of N-terminal sequencing in scientific research and biopharmaceutical development. Therefore, we have established anautomated Edman sequencing platform, complemented by comprehensive purification, quantification, and quality control systems, providing customers with one-stop N-terminal sequence analysis services.

Our advantages include:

• High-sensitivity equipment: Equipped with the advanced PPSQ-51A sequencer, capable of identifying protein samples as small as 1~5 pmol

• Purification support services: Offering pre-processing solutions such as SDS-PAGE band recovery and PVDF membrane transfer

• Multi-cycle control: Capable of sequencing up to 30 amino acid residues, accurately reconstructing the N-terminal structure

• Detailed report delivery: Including original chromatograms, electrophoresis images, sequence alignment, and technical notes

 

In today's context of emphasizing 'precise sequencing and full-chain validation', Edman degradation has not been phased out but has instead returned to the forefront in the development of various new biological products. Edman degradation sequencing still holds unique value in the field of protein research due to its chemical analysis certainty and specificity for N-terminal analysis. It not only complements mass spectrometry technology but also plays an irreplaceable role in certain critical application scenarios.BioPark Biotechprovides professional protein N-terminal sequence analysis services based on Edman degradation, capable of solving your project issues, accelerating your research projects, and offering you a high-quality service experience.

 

BioPark Biotech—Characterization of biological products, a premium service provider for multi-omics biological mass spectrometry

 

Related services:

Protein N-terminal sequence analysis based on Edman degradation