Mechanism of N/C Terminal Sequencing

N- and C-terminal sequencing are fundamental techniques in molecular biology, offering critical insights into the structure, function, and integrity of proteins. These sequencing methods are pivotal for understanding the full landscape of proteomics and ensuring the quality of biopharmaceutical products.

 

Mechanism of N-Terminal Sequencing

1. Edman Degradation

Edman degradation is a classical method used for N-terminal sequencing. The process involves the stepwise removal of amino acids from the N-terminus of a protein or peptide. Here's how it works:

 

(1) Phenyl Isothiocyanate (PITC) Reaction

The N-terminal amino acid reacts with PITC, forming a phenylthiocarbamoyl derivative.

 

(2) Acidic Cleavage

This derivative is cleaved under acidic conditions, yielding an anilinothiazolinone (ATZ) amino acid.

 

(3) Conversion and Identification

The ATZ amino acid is converted to a more stable phenylthiohydantoin (PTH) derivative, which can be identified using chromatography.

 

2. Mass Spectrometry (MS)

Mass spectrometry complements Edman degradation by addressing its limitations. The MS method for N-terminal sequencing involves:

 

(1) Ionization

The protein sample is ionized, typically using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI).

 

(2) Mass Analysis

The ionized particles are analyzed based on their mass-to-charge ratio (m/z).

 

(3) Sequence Determination

The data is interpreted to determine the sequence of the N-terminal amino acids and identify any modifications.

 

Mechanism of C-Terminal Sequencing

1. Carboxypeptidase Digestion

Carboxypeptidase digestion is a common method for C-terminal sequencing. This enzymatic approach involves:

 

(1) Enzyme Action

Carboxypeptidases sequentially cleave amino acids from the C-terminus of the protein or peptide.

 

(2) Amino Acid Identification

The released amino acids are identified using chromatography or mass spectrometry.

 

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

LC-MS/MS is a powerful technique for C-terminal sequencing. The process includes:

 

(1) Liquid Chromatography (LC)

Proteins are separated based on their properties using liquid chromatography.

 

(2) Mass Spectrometry (MS/MS)

The separated proteins are ionized and fragmented, and the fragments are analyzed to determine the C-terminal sequence.

 

Applications and Significance

1. Biopharmaceutical Quality Control

Ensuring the integrity and functionality of biopharmaceutical products, such as therapeutic proteins, antibodies, and vaccines, is critical. N/C terminal sequencing verifies that these products are correctly synthesized and free from truncations or undesirable modifications, ensuring efficacy and safety.

 

2. Proteomics Research

In proteomics, N/C terminal sequencing aids in identifying and characterizing proteins within complex biological samples. It helps researchers understand protein function, interaction, and cellular pathways, leading to discoveries of new proteins and their roles.

 

3. Post-Translational Modifications

PTMs are crucial for 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.

 

By combining techniques like Edman degradation and mass spectrometry, researchers can achieve comprehensive and accurate analyses of protein terminal sequences. These methods are indispensable for advancing our understanding of protein biology, ensuring the quality of biopharmaceuticals, and uncovering the complexities of proteomics. Through meticulous execution and innovative approaches, N/C terminal sequencing continues to be a cornerstone of molecular biology. MtoZ Biolabs provides integrate N/C terminal sequencing service.