De Novo Protein Sequencing: Basic Concepts and Applications

Protein de novo sequencing refers to the direct determination of the amino acid sequence of a protein without relying on known protein databases. Unlike other analytical methods that rely on known protein sequence databases or known mass spectrometry databases. The software developed by this method includes PepNovo, PEAKS, and pNovo. De novo sequencing uses tandem mass spectrometry to directly analyze the fragment characteristics of peptides. Therefore, peptide sequences not included in the protein database, protein sequences of new species, and protein sequences that have not yet been sequenced in the genome can be analyzed.

 

Basic Principles of De Novo Sequencing

In mass spectrometry analysis, the particles of the substance to be analyzed are ionized before passing through the electromagnetic field. Because. In the process of peptide identification using tandem mass spectrometry, peptide de novo sequencing can infer the most likely peptide sequence directly from the secondary spectra. After finding a specific fracture pattern, the corresponding amino acid information can be calculated based on the mass difference between the mass spectrometry peak and the post-translational modification of the amino acid. Ions with different mass-to-charge ratios are subjected to different forces in the electromagnetic field, and their trajectories and flight times are different, so they can separate and detect the characteristics of ions.

 

Applications of De Novo Sequencing

1. Analysis and Detection of Unknown Peptide Sequences in Biological Samples

2. Detection of Peptides with Missing Terminal Residues

3. Identification of Lysine and Leucine

4. Identification of N-Terminus Capped and Cyclic Proteins

5. Accurate Determination of the Sequence Information of Unknown Proteins or Peptides for Easy Analysis

6. Functional Study of Unknown Proteins

7. Accurate Determination of Commercial Modified Protein and Enzyme Sequences

8. Accurate Determination of the Sequence of Proteins Expressed in Stable Cell Lines

9. Monoclonal Analysis of the Primary Structure Sequence of Antibodies etc.

 

De novo protein sequencing is more complex and challenging than sequencing methods that rely on databases. Short polypeptide fragments are relatively easy to sequence, but as the fragment length increases, the possible amino acid combinations increase exponentially, increasing the complexity of the analysis.