Edman Degradation vs Full-Length Sequencing: A Comprehensive Analysis Strategy from N-Terminus to C-Terminus

In the research of protein function, recombinant protein validation, and antibody drug development, precise confirmation of primary structure (i.e., the linear sequence of amino acids) is the most fundamental and crucial step. To achieve this goal, researchers often use two technical approaches:

• Edman degradation: A classic chemical method, focusing on sequential analysis of N-terminal amino acids;

• Protein full-length de novo sequencing: Based on high-resolution mass spectrometry and AI algorithms, it can restore the complete protein sequence from N-terminal to C-terminal.

These two techniques each have advantages and limitations. So, in actual projects, how should they be combined? Is there a more efficient, comprehensive N-terminal+C-terminal integrated structural analysis strategy? This article will systematically review the collaborative application value of Edman degradation and full-length sequencing from the aspects of technical principles, applicable scenarios, data complementarity, and strategic recommendations.

 

I. Edman Degradation: Precise, Visual N-terminal Sequence Reading Method

1. Principle Review

Edman degradation selectively labels the free N-terminal α-amino acid of the protein using phenyl isothiocyanate (PITC) and removes and identifies one residue per cycle. This method has extremely high credibility for the 'stepwise confirmation' of the amino acid sequence.

 

2. Technical Advantages

(1) High precision: Each round of sequence results is confirmed based on chemical reactions;

(2) Low background: Suitable for single-band, high-purity proteins;

(3) Does not rely on databases or mass spectrometry algorithms, results are intuitive;

(4) Strong ability to identify starting residues, can determine N-terminal modifications/deletions/mutations.

 

3. Limitations

(1) Limited to free N-terminal proteins (ineffective if N-terminal is modified or blocked);

(2) Unable to recognize internal protein structures;

(3) Generally limited sequencing depth to within 10–15 amino acids;

(4) Requires high protein purity and a sample amount of no less than 5–10 pmol.

 

II. Full-Length Sequencing: Restoring Full Protein Structure from Spectral Fragments

1. Principle Summary

Protein full-length sequencing uses various proteases (Trypsin, Glu-C, Asp-N, etc.) to cleave the target protein, generating a series of overlapping peptides. After high-resolution mass spectrometry detection, AI assembly algorithms (such as PEAKS, pNovo, DeepNovo, etc.) directly restore the complete amino acid sequence of the protein from the original spectrum without reference to a database.

 

2. Technical Advantages

(1) Does not rely on known sequences, suitable for unknown proteins, mutants, artificial constructs;

(2) Can identify single-point mutations, isomeric mixtures, splice variants;

(3) Simultaneously analyzes N-terminal, C-terminal, internal structures, and post-translational modifications (PTMs);

(4) Starting amount can be as low as 1–10 ng, suitable for low-abundance samples.

 

3. Limitations

(1) N-terminal analysis depends on peptide generation, may miss starting residues;

(2) When C-terminal peptides are large or have diverse modifications, identification difficulty increases;

(3) Results depend on spectrum quality and assembly algorithms;

(4) Data analysis is more complex, requires manual review and algorithmic joint judgment.

 

III. Recommended Typical Application Strategies

📌 Scenario 1: Recombinant Antibody Sequencing Validation

• Pain point: Does the expressed product retain the starting residue? Are there minor mutations?

• Recommended strategy: Use Edman for precise N-terminal positioning of light/heavy chains, full-length sequencing to confirm V region structure and C-terminal construct integrity.

 

📌 Scenario 2: Fusion Protein Functional Domain Splicing Confirmation

• Pain point: Is the connection region fully expressed? Is there an extension or tag loss at the C-terminal?

• Recommended strategy: Full-length sequencing to splice the entire structure, Edman to confirm starting residues and cleavage positions.

 

📌 Scenario 3: Unknown Protein Primary Structure Analysis

• Pain point: No database matches, no nucleic acid sequence for reference

• Recommended strategy: Full-length sequencing for main structure analysis + Edman to strengthen N-terminal results, achieving database-independent identification.

 

IV. Biotech Pack's Integrated Structural Analysis Solution

Biotech Pack relies on high-resolution mass spectrometry platforms (Orbitrap Eclipse, timsTOF Pro) and automated Edman sequencers to provide the following service combinations:

• Edman sequencing service: Supports direct sample application on PVDF membrane, minimum detection amount 1 pmol, sequence depth up to 15 residues;

• Protein full-length sequencing service: Multi-enzyme cleavage + AI algorithm assembly + manual review, supports modification identification, isomer analysis;

• Integrated structural report output: Includes full sequence map, mutation annotations, modification sites, terminal confirmation information, meeting the needs for biologic drug registration and IND application.

 

We are committed to helping researchers and pharmaceutical companies move from 'seeing sequences' to 'understanding structures,' bridging the closed-loop expression verification path of proteins from N-terminal to C-terminal. In protein structural validation tasks, no single technique can 'cover everything.' Only by reasonably combining multiple techniques can higher resolution and higher credibility of structural panorama be obtained.

 

Biotech Pack Biotechnology -- Characterization of Biologics, Quality Mass Spectrometry Detection Service Provider for Multi-Omics

 

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