Workflow of Protein Primary Structure Characterization

Protein primary structure refers to the linear sequence of amino acids in a protein molecule, which determines the protein's three-dimensional structure and function. Therefore, accurately characterizing the primary structure of proteins is crucial for understanding their function and mechanism. The following is the standard workflow for protein primary structure characterization:

 

Sample Preparation

Before characterizing the primary structure of proteins, it is necessary to extract proteins from biological samples. Sample preparation involves cell lysis, protein extraction, and purification.

 

1. Cell Lysis

Cell lysis involves disrupting the cell membrane using physical or chemical methods to release intracellular proteins. Common methods include sonication, freeze-thaw cycles, and the use of lysis buffers.

 

2. Protein Extraction

Protein extraction entails separating proteins from the cell lysate. This typically requires techniques such as centrifugation, filtration, and precipitation.

 

3. Protein Purification

Protein purification separates and purifies the target protein using various biochemical techniques such as gel filtration chromatography, ion exchange chromatography, and affinity chromatography for subsequent analysis.

 

Protein Hydrolysis

To analyze the primary structure of proteins, the protein molecules need to be cleaved into smaller peptides. This is usually achieved by using specific proteases like trypsin.

 

1. Enzymatic Digestion

Appropriate enzymes and reaction conditions are selected to cleave the protein at specific sites. Commonly used enzymes include trypsin, chymotrypsin, and Staphylococcus aureus protease.

 

2. Purification of Digestion Products

After enzymatic digestion, the generated peptides need to be purified using techniques such as high-performance liquid chromatography (HPLC).

 

Mass Spectrometry Analysis

Mass spectrometry analysis is the most critical step in protein primary structure characterization, where the mass of the peptides is measured to infer their amino acid sequences.

 

1. Sample Ionization

The purified peptide samples are ionized into gas-phase ions using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) techniques.

 

2. Mass Spectrometry Measurement

The ionized peptides are analyzed by a mass spectrometer to obtain mass spectra. Commonly used mass spectrometry techniques include tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry.

 

3. Data Analysis

Software for mass spectrometry data analysis is employed to interpret the mass spectra, deducing the amino acid sequences of the peptides, and confirming the identity and primary structure of the proteins through database searches.

 

Data Validation

To ensure the accuracy of the results, multiple repeat experiments are usually conducted, and the amino acid sequences are validated using other techniques such as Edman degradation.

 

MtoZ Biolabs provides integrate protein primary structure characterization service.