Principle of Protein Sequencing Methods

Proteins are essential molecules that perform various functions in living organisms, with their structure being closely linked to their function. Understanding the amino acid sequences of proteins is crucial for studying their functions and roles in biological processes. The primary methods for protein sequencing are chemical degradation and mass spectrometry.

 

Chemical Degradation Methods

1. Edman Degradation

Edman degradation is a classical chemical method used to sequentially remove and identify amino acids from the N-terminus of a protein. The process includes:

 

(1) Coupling Reaction

Under alkaline conditions, phenyl isothiocyanate (PITC) reacts with the N-terminal amino acid of the protein to form a phenylthiocarbamoyl derivative.

 

(2) Cyclization Reaction

Under acidic conditions, this derivative undergoes cyclization to produce a phenylthiohydantoin (PTH)-amino acid, exposing the new N-terminal amino acid.

 

(3) Identification

The PTH-amino acid is analyzed by high-performance liquid chromatography (HPLC) to determine its identity.

 

This method allows for stepwise identification of amino acids in a protein sequence. However, its efficiency is limited for longer peptide segments and proteins with modified or blocked N-termini.

 

2. Limitations of Chemical Degradation

Despite its significance, Edman degradation has limitations. It is ineffective for proteins with modified or blocked N-termini and its efficiency decreases with longer peptide chains, often necessitating complementary methods for comprehensive analysis.

 

Mass Spectrometry Methods

Mass spectrometry (MS) identifies and quantifies substances by measuring the mass-to-charge ratio (m/z) of ions. In protein sequencing, MS involves:

 

1. Sample Ionization

Converting the protein sample into gas-phase ions using techniques like matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI).

 

2. Mass Analysis

Separating the generated ions based on their m/z ratio using mass analyzers such as time-of-flight (TOF), ion trap, and quadrupole analyzers.

 

3. Data Analysis

Interpreting the peak information in the mass spectrum to deduce the protein's amino acid sequence.

 

Tandem MS (MS/MS)

Tandem mass spectrometry (MS/MS) provides detailed sequence information through:

 

1. Primary MS

Obtaining the parent ion of the protein.

 

2. Fragmentation

Inducing fragmentation of selected parent ions using collision-induced dissociation (CID) or other methods.

 

3. Secondary MS

Analyzing fragment ions to obtain their m/z ratios, thereby deducing the original molecule's structure and sequence.

 

Chemical degradation and mass spectrometry each have their strengths: chemical degradation is suitable for analyzing short peptide sequences, while mass spectrometry excels in high-throughput and complex sample analysis. As technology advances, these methods will continue to drive progress in protein science. MtoZ Biolabs provides integrate protein sequencing service.