Mechanism of De Novo Protein Sequencing

De Novo Protein Sequencing is a method for protein sequence analysis that does not rely on existing databases.

 

Sample Preparation

1. Protein Extraction

Extract protein samples from cells or tissues, ensuring the samples are pure and free from contamination.

 

2. Protein Digestion

Use enzymes (such as trypsin) to cut the proteins into smaller peptides for subsequent mass spectrometry analysis.

 

3. Sample Purification

Purify the peptides using liquid chromatography to remove impurities and ensure the accuracy of mass spectrometry analysis.

 

Mass Spectrometry Analysis

1. Peptide Ionization

Convert peptides into gas-phase ions using electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI).

 

2. Mass-to-Charge Ratio Measurement

Measure the mass-to-charge ratio (m/z) of the peptide ions in the mass spectrometer to obtain a series of precursor ion spectra.

 

3. Peptide Fragmentation

Further fragment the precursor ions using methods such as collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or electron transfer dissociation (ETD) to generate fragment ion spectra.

 

Data Processing

1. Spectrum Interpretation

Use specialized software (such as PEAKS or Byonic) to interpret the mass spectrometry data and identify the signals of precursor and fragment ions.

 

2. Sequence Reconstruction

Deduce the amino acid sequences of the peptides based on the fragment ion spectra using algorithms, and gradually piece together the complete protein sequence.

 

3. Result Validation

Verify the accuracy of the sequencing results using methods such as reverse sequence confirmation and isotope labeling.

 

Mechanism Analysis

1. Fragmentation Mechanism

By choosing appropriate fragmentation methods, stable and easily identifiable fragment ions are produced, enhancing the readability of mass spectra.

 

2. Data Interpretation Algorithms

Advanced data interpretation algorithms can efficiently process large-scale mass spectrometry data and accurately identify amino acid sequences.

 

3. Multiple Enzyme Digestion Strategy

Employing multiple enzyme digestion strategies can increase sequence coverage, ensuring each amino acid position is verified multiple times.

 

De novo protein sequencing achieves accurate decoding of unknown protein sequences through precise sample preparation, advanced mass spectrometry analysis, and efficient data processing. MtoZ Biolabs provides integrate De Novo protein sequencing service.