Mechanism of Protein Full-Length Sequencing

Proteins are essential molecules that execute a variety of functions within organisms, including catalysis, structural support, and regulation. Precisely determining the amino acid sequences of proteins is critical for studying their structure and function. Full-length protein sequencing involves multiple methods and steps to analyze complex protein samples comprehensively.

 

Sample Preparation

The initial step in full-length protein sequencing is sample preparation, which involves protein extraction, purification, and quantification. Common procedures include:

 

1. Protein Extraction

Proteins are extracted from cells or tissues, typically using lysis buffers to break down cellular structures.

 

2. Protein Purification

Target proteins are separated and purified using chromatographic techniques such as ion exchange chromatography, affinity chromatography, and gel filtration chromatography.

 

3. Protein Quantification

The protein content is quantified using methods like the Bradford assay or the BCA assay to ensure there is sufficient protein for sequencing.

 

Protein Degradation

To determine the full-length sequences of proteins, they must be degraded into smaller peptides. Common methods include:

 

1. Chemical Degradation

Edman degradation sequentially removes and identifies each amino acid to determine the sequence.

 

2. Enzymatic Digestion

Specific proteases (e.g., trypsin, chymotrypsin) cleave proteins into peptides suitable for mass spectrometry analysis.

 

Peptide Separation

The peptides generated from degradation need to be further separated for subsequent analysis. Common methods include:

 

1. Liquid Chromatography

High-performance liquid chromatography (HPLC) separates peptides based on their hydrophilicity or hydrophobicity.

 

2. Electrophoresis

Two-dimensional electrophoresis (2-DE) separates peptides based on their isoelectric points and molecular weights.

 

Mass Spectrometry Analysis

Mass spectrometry (MS) is the core step in full-length protein sequencing. It measures the mass and determines the amino acid sequence of peptides. Common MS techniques include:

 

1. Electrospray Ionization Mass Spectrometry (ESI-MS)

Suitable for analyzing larger biomolecules.

 

2. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)

Offers high sensitivity and resolution, making it ideal for proteomics studies.

 

Data Analysis

The data generated from mass spectrometry require complex bioinformatics analysis to reconstruct the full-length protein sequence. Key steps include:

 

1. Peptide Identification

Mass spectrometry data are matched with known protein sequences using database search algorithms (e.g., Mascot, Sequest).

 

2. Sequence Assembly

Identified peptide sequences are assembled into a complete protein sequence.

 

3. Result Validation

The accuracy of assembled sequences is confirmed through biological experiments such as Western blotting and further mass spectrometry validation.

 

Full-length protein sequencing has extensive applications, including the discovery of new proteins, identification of disease biomarkers, and study of drug targets. Future advancements in technology will further enhance the role of full-length protein sequencing in driving biomedical research development. MtoZ Biolabs provides integrate protein full-length sequencing service.