Workflow of N/C Terminal Sequencing

N- and C-terminal sequencing are essential techniques in molecular biology, providing crucial insights into the structure and function of proteins. These sequencing methods are indispensable for studying protein integrity, identifying post-translational modifications (PTMs), and ensuring the quality of biopharmaceutical products.

 

Workflow of N-Terminal Sequencing

1. Sample Preparation

The initial step in N-terminal sequencing involves meticulous sample preparation. Protein samples can be in various formats, such as gel bands, gel spots, or liquid samples. For Edman degradation, proteins are typically separated by SDS-PAGE and blotted onto PVDF membranes. The purity of the samples is crucial, as contaminants can interfere with the sequencing process.

 

2. Edman Degradation

Edman degradation is a classical method for N-terminal sequencing. It sequentially removes one amino acid at a time from the N-terminus, which is then identified by chromatography. This method is highly accurate but has limitations with blocked N-termini or extensively modified proteins.

 

3. Mass Spectrometry (MS)

To overcome the limitations of Edman degradation, mass spectrometry is employed. MS can identify peptides and proteins by measuring the mass-to-charge ratio of ionized particles. This technique is particularly useful for detecting N-terminal blockages and PTMs, providing a comprehensive analysis of the protein's N-terminus.

 

4. Data Analysis

The sequences obtained from Edman degradation and MS are analyzed using bioinformatics tools. These tools help identify the amino acid sequences and any modifications present, ensuring accurate interpretation of the protein's N-terminus.

 

Workflow of C-Terminal Sequencing

1. Sample Preparation

Similar to N-terminal sequencing, the preparation of samples for C-terminal sequencing requires high purity and careful handling to avoid contamination. Samples can be prepared in gel or liquid forms, with stringent requirements for purity and concentration.

 

2. Carboxypeptidase Digestion

One common method for C-terminal sequencing is carboxypeptidase digestion. This enzymatic process sequentially cleaves amino acids from the C-terminus, allowing for their identification. While effective for unmodified proteins, this method is less suitable for proteins with C-terminal modifications.

 

3. Tandem Mass Spectrometry (LC-MS/MS)

LC-MS/MS is a powerful technique for C-terminal sequencing. It combines liquid chromatography with mass spectrometry to provide a detailed analysis of the C-terminal sequence and associated modifications. This method offers high sensitivity and accuracy, making it ideal for studying complex proteins.

 

4. Data Analysis

The data from carboxypeptidase digestion and LC-MS/MS are processed using advanced bioinformatics tools. These tools enable the identification of the C-terminal amino acid sequence and any PTMs, providing a comprehensive understanding of the protein's C-terminus.

 

Applications of N/C Terminal Sequencing

1. Biopharmaceutical Quality Control

N/C terminal sequencing is crucial for verifying the integrity and functionality of biopharmaceutical products, such as therapeutic proteins, antibodies, and vaccines. Accurate terminal sequencing ensures that these products meet quality standards and function as intended.

 

2. Proteomics Research

In proteomics, N/C terminal sequencing helps researchers understand protein function, interaction, and cellular pathways. By determining the terminal sequences, scientists can gain insights into how proteins work and how they are regulated within the cell.

 

3. Post-Translational Modifications

PTMs play a vital role in protein function and stability. N/C terminal sequencing allows for the identification and characterization of PTMs, providing essential information on how these modifications affect protein activity.

 

The workflow of N/C terminal sequencing involves several critical steps, from sample preparation to data analysis. By combining methods such as Edman degradation, carboxypeptidase digestion, and mass spectrometry, researchers can achieve a comprehensive and accurate analysis of protein terminal sequences. These techniques are indispensable for advancing our understanding of protein biology and ensuring the quality of biopharmaceutical products. MtoZ Biolabs provides integrate N/C terminal sequencing service.