Principle of N/C Terminal Sequencing

N- and C-terminal sequencing are fundamental techniques in molecular biology, offering critical insights into protein structure and function. These methods are essential for studying protein integrity, understanding biological processes, and ensuring the quality of biopharmaceutical products.

 

Principles of N/C Terminal Sequencing

1. N-Terminal Sequencing

The N-terminus of a protein is the starting point of protein synthesis and plays a crucial role in determining its subcellular localization, degradation rate, and overall stability. N-terminal sequencing involves determining the sequence of amino acids from the amino terminus of the protein. Two primary methods are used for this purpose:

 

(1) Edman Degradation

① Principle: Edman degradation sequentially removes one amino acid at a time from the N-terminus, which is then identified by chromatography.

② Advantages: High accuracy in determining the N-terminal sequence of proteins.

③ Disadvantages: Inefficient for proteins with blocked N-termini or extensive post-translational modifications (PTMs).

 

(2) Mass Spectrometry (MS)

① Principle: MS can identify peptides and proteins by measuring the mass-to-charge ratio of ionized particles.

② Advantages: Effective in identifying N-terminal blockages and PTMs.

③ Disadvantages: Requires sophisticated instrumentation and expertise.

 

2. C-Terminal Sequencing

The C-terminus, being the end point of protein synthesis, is vital for protein function and interaction with other biomolecules. C-terminal sequencing identifies the sequence of amino acids from the carboxyl terminus. Major methods include:

 

(1) Carboxypeptidase Digestion

① Principle: Enzymatic digestion with carboxypeptidases sequentially cleaves amino acids from the C-terminus.

② Advantages: Simple and effective for unmodified proteins.

③ Disadvantages: Less effective for proteins with C-terminal modifications.

 

(2) Tandem Mass Spectrometry (LC-MS/MS)

① Principle: Combines liquid chromatography with mass spectrometry to analyze the C-terminal sequence.

② Advantages: High sensitivity and capability to analyze PTMs.

③ Disadvantages: High operational costs and technical complexity.

 

Workflow of N/C Terminal Sequencing

The workflow for N/C terminal sequencing typically involves the following steps:

 

1. Sample Preparation

Protein samples are prepared in appropriate formats such as gel bands, gel spots, or liquid samples with specific purity and concentration requirements.

 

2. Sequencing

(1) For N-terminal sequencing, samples may undergo Edman degradation or MS analysis.

(2) For C-terminal sequencing, samples may be treated with carboxypeptidases or analyzed using LC-MS/MS.

 

3. Data Analysis

The resulting sequences are analyzed using bioinformatics tools to identify amino acid sequences and PTMs.

 

4. Reporting

Comprehensive reports are generated, including experimental procedures, chromatographic and mass spectrometric parameters, raw data files, and identified sequences.

 

Applications of N/C Terminal Sequencing

1. Biopharmaceutical Quality Control

Ensuring the integrity and functionality of therapeutic proteins, antibodies, and vaccines by verifying their terminal sequences and identifying any modifications or truncations.

 

2. Proteomics Research

Understanding protein function, interaction, and cellular pathways by analyzing terminal sequences.

 

3. Post-Translational Modifications

Identifying and characterizing PTMs, which are crucial for protein activity, stability, and interaction.

 

N/C terminal sequencing is a vital tool in molecular biology, providing detailed insights into protein structure and function. By combining various sequencing methods, such as Edman degradation and mass spectrometry, researchers can overcome the limitations of individual techniques and achieve accurate and comprehensive protein analysis. These methodologies are indispensable for advancing proteomics research, ensuring biopharmaceutical quality, and unraveling the complexities of protein biology. MtoZ Biolabs provides integrate N/C terminal sequencing service.