Mechanism of Peptide Sequencing

Peptide sequencing is a crucial technique in protein research, allowing scientists to understand the structure and function of proteins by determining the order of amino acids in a peptide chain.

 

The fundamental principle of peptide sequencing techniques is to break down peptide chains into smaller, analyzable fragments using chemical or enzymatic methods, and then detect these fragments to determine the order of amino acids. The main methods of peptide sequencing include the Edman degradation method and mass spectrometry.

 

Edman Degradation Method

The Edman degradation method is a classic peptide sequencing technique that determines the amino acid sequence by progressively cleaving the terminal amino acid of the peptide chain and identifying it. The specific steps are as follows:

 

1. PITC Reaction

Under alkaline conditions, the N-terminal amino acid of the peptide chain reacts with phenylisothiocyanate (PITC) to form a phenylthiocarbamoyl derivative.

 

2. Cyclization and Cleavage

Under acidic conditions, this derivative cyclizes and cleaves from the peptide chain, forming a phenylthiohydantoin (PTH)-amino acid.

 

3. Identification

The PTH-amino acid is identified using high-performance liquid chromatography (HPLC) or other analytical techniques.

 

4. Repeat Process

The above steps are repeated to identify each amino acid in the peptide chain sequentially.

 

Mass Spectrometry

Mass spectrometry is a widely used peptide sequencing technique with high sensitivity and high throughput. The main steps of mass spectrometry are as follows:

 

1. Sample Ionization

Peptide molecules are ionized using techniques such as electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI).

 

2. Mass Analysis

The ionized peptide molecules are separated in a mass spectrometer and detected based on their mass-to-charge ratio (m/z).

 

3. Peptide Fragmentation

Peptide molecules are further fragmented using methods such as collision-induced dissociation (CID), generating a series of fragment ions with specific m/z values.

 

4. Data Analysis

Software for mass spectrometry data analysis matches and reconstructs the m/z of fragment ions to determine the amino acid sequence of the peptide chain.

 

Data Interpretation and Applications

The amino acid sequence data obtained through Edman degradation and mass spectrometry can be used in several areas:

 

1. Protein Function Research

Understanding the amino acid sequence of a protein helps elucidate its biological function and mechanism of action.

 

2. Protein Structure Analysis

The amino acid sequence is the basis for studying the tertiary and quaternary structures of proteins.

 

3. Disease Diagnosis and Treatment

Peptide sequencing of disease-related proteins can identify potential biomarkers and therapeutic targets.