Explore De Novo Protein Sequencing

De novo protein sequencing refers to the method of deducing the amino acid sequence of a protein or peptide directly from experimental data, without relying on known DNA or protein database information. It is useful for studying proteins in species without reference sequences, or for exploring new variants and modifications of proteins.

 

Technical Principle

De novo sequencing usually relies on mass spectrometry (MS) technology, especially tandem mass spectrometry (MS/MS), to determine the sequence of amino acids by measuring the mass differences of peptide molecules under specific fragmentation. In MS/MS experiments, peptide ions are selectively subjected to collision-induced dissociation (CID), electron transfer dissociation (ETD), or electron capture dissociation (ECD), generating a series of fragment ions, the mass differences of which are used to infer the amino acid sequence of the original peptide.

 

Application Scenarios

De novo sequencing is very useful for studying the protein expression of species that have not yet been sequenced, or when the known sequence database is insufficient to cover all proteins in a sample. It can also be used to identify unknown variants and isoforms of proteins, or to detect post-translational modifications.

 

Technical Challenges

Determining the accurate amino acid sequence requires high-precision and high-resolution mass spectrometry data. Moreover, the complexity of sequence inference increases significantly with the length of the peptide chain. In addition, some amino acids, such as leucine and isoleucine, have the same mass in mass spectrometry, so they cannot be distinguished without additional information.

 

Software Tools

Processing mass spectrometry data and deriving amino acid sequences from it requires specialized software and algorithms. There are several software packages available for de novo protein sequencing, such as PEAKS, Novor, and DeepNovo.

 

Although de novo protein sequencing is technically challenging, its application scope and accuracy have significantly improved with the continuous development of mass spectrometry technology and data analysis algorithms, making it an important tool in proteomic research.