Principle of Unknown Proteins Sequencing

Proteins are essential molecules in living organisms that perform a wide range of functions. They are composed of chains of amino acids and exhibit highly complex three-dimensional structures. The function of a protein is determined by its specific amino acid sequence and spatial conformation. Unknown protein sequencing is an important biological research technique, mainly involving methods such as mass spectrometry (MS) and tandem mass spectrometry (MS/MS).

 

Mass Spectrometry (MS)

Mass spectrometry is a technique that measures the mass-to-charge ratio (m/z) of molecules to determine their molecular mass. It ionizes the sample and then measures the m/z value of the ions to determine the mass of the protein or peptide.

 

1. Ionization Methods

The first step in mass spectrometry is ionizing the protein or peptide. Common ionization techniques include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). ESI is suitable for liquid samples and can produce multiply charged ions, while MALDI is suitable for solid samples and typically produces singly charged ions.

 

2. Mass Analysis

The ionized molecules are sent to a mass analyzer for separation. Common mass analyzers include Quadrupole, Time-of-Flight (TOF), and Fourier Transform Ion Cyclotron Resonance (FT-ICR). These analyzers separate ions based on their m/z value and generate a mass spectrum.

 

3. Data Analysis

In the mass spectrum, each peak corresponds to a specific m/z value. By analyzing these peaks, the mass of the protein or peptide can be determined. Combined with database searches, the amino acid sequence of the unknown protein can be inferred.

 

Tandem Mass Spectrometry (MS/MS)

Tandem mass spectrometry (MS/MS) further improves the accuracy of protein sequencing. It involves two stages of mass analysis, providing more detailed structural information.

 

1. First Mass Spectrometry (MS1)

In MS/MS, the first mass spectrometry (MS1) analysis measures the overall mass of the protein or peptide. Specific ions (precursor ions) are then selected for the next stage.

 

2. Ion Fragmentation

Before entering the second mass spectrometry (MS2) analysis, the precursor ions are fragmented into smaller ions (product ions) through methods such as Collision-Induced Dissociation (CID) or Electron Transfer Dissociation (ETD).

 

3. Second Mass Spectrometry (MS2)

In MS2 analysis, the m/z values of these product ions are measured. By analyzing the mass spectrum of the product ions, the amino acid sequence of the precursor ion can be determined.

 

4. Data Integration

Combining the data from MS1 and MS2 allows for a more accurate inference of the amino acid sequence of the unknown protein. Database searches and algorithmic analysis provide comprehensive structural information about the protein.

 

Mass spectrometry and tandem mass spectrometry are vital tools in modern proteomics research. They are used not only for sequencing unknown proteins but also for studying protein modifications, protein interactions, and disease biomarker discovery. With continuous technological advancements, the accuracy and efficiency of protein sequencing will improve, providing stronger support for biomedical research. MtoZ Biolabs provides integrate unknown proteins sequencing service.