Edman Degradation vs Mass Spectrometry: A Comparison of Protein Sequencing Techniques (Part II)

Proteins serve as the core executors of cellular functions, and their amino acid sequence information not only reveals their structure and function but also provides the basis for applications such as biomarker discovery, antibody identification, and vaccine design. Protein sequencing, a crucial technology for analyzing the composition and arrangement of amino acids, has developed over several decades into two main approaches: Edman degradation and Mass Spectrometry (MS). What are the advantages of these two protein sequencing techniques, and in which research scenarios are they applicable? This article will provide a comprehensive comparison and analysis.

 

What is Edman degradation?

Edman degradation is a classic protein sequencing technique developed by Pehr Edman in 1950. The principle involves chemically cleaving amino acids one by one from the N-terminus and identifying them, thereby inferring the protein sequence step by step.

 

Core Mechanism

(1) PITC Labeling: Phenyl isothiocyanate (PITC) reacts with the N-terminal amino acid to form a cyclic derivative.

(2) Acid Cleavage: Releases the first amino acid derivative (PTH-aa).

(3) High-Performance Liquid Chromatography (HPLC) Analysis: Identifies the type of amino acid.

(4) Repeat the above steps to sequentially read amino acids.

 

Advantages

(1) High fidelity, suitable for known sequences or antibody validation.

(2) Suitable for highly purified proteins.

(3) Insensitive to modifications in the amino acid sequence.

 

Limitations

(1) Typically only determines the first 30–50 amino acids.

(2) Requires high purity and large quantities of samples.

(3) Ineffective for C-terminal and middle sequences.

(4) Cannot identify post-translational modifications (PTMs).

 

What is Mass Spectrometry Protein Sequencing?

Mass spectrometry sequencing is a modern technological approach that involves accurately measuring the mass-to-charge ratio (m/z) of proteins or peptides and using database searches or de novo algorithms to decipher the protein sequence.

 

Core Process

（1）Protease Digestion: Proteins are hydrolyzed into peptides, such as by trypsin.

(2) LC-MS/MS Analysis: Liquid chromatography separation followed by tandem mass spectrometry fragmentation of peptides.

(3) Database matching or de novo algorithm to infer sequences.

 

Advantages

（1）High throughput: Can simultaneously determine thousands of proteins.

(2) High sensitivity: Can sequence samples at nanogram or even picogram levels.

(3) Identifies post-translational modifications: such as phosphorylation, acetylation, glycosylation, etc.

(4) Suitable for complex samples: such as cell lysates, tissue extracts, plasma, etc.

 

Limitations

(1) Difficult to analyze small peptides (< 5 amino acids).

(2) De novo algorithm results require validation and have some uncertainty.

(3) Complex modifications and isomers may increase identification difficulty.

 

Comparison Overview

 

Features	Edman Degradation	Mass Spectrometry Sequencing
Principle	Chemical cleavage + chromatography analysis	Peptide fragmentation + mass spectrometry analysis
Throughput	Single protein	High throughput parallel analysis
Sensitivity	Microgram level	Nanogram level or even lower
Modification Recognition	Not supported	Can recognize various PTMs
Data Dependency	Database not required	Requires database or algorithm support
Cost and time	High cost, long duration	Low cost, short cycle
Application scenarios	Verification after protein purification, antibody sequence analysis	Proteomics analysis, unknown sequence determination, post-translational modification research

 

IV. Recommendations for protein sequencing technology selection

If you have a highly purified single protein and wish to validate its N-terminal sequence or antibody fragment, Edman degradation remains valuable;

If you are interested in the overall protein expression profile, post-translational modifications, or unknown protein sequence determination, mass spectrometry is a more modern and efficient choice.

 

In today's growing demand for protein sequencing, choosing a reliable experimental platform is crucial. Biotech Pack BioTech leverages advanced Orbitrap and Q-TOF mass spectrometry platforms to offer a diverse range of services including N/C terminal sequencing, de novo peptide sequencing, and post-translational modification analysis. We not only meet the basic needs of researchers but also help biopharmaceutical companies advance antibody drug development, vaccine validation, and innovative protein design. Whether you are a research institution, hospital researcher, or biopharmaceutical company, feel free to contact us for one-stop solutions to protein sequencing challenges.

 

Biotech Pack BioTech--Characterization of biological products, high-quality service provider for multi-group biological mass spectrometry detection

 

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