Principle of De Novo Protein Sequencing

Generally, there are two methods for protein sequence analysis: database search and de novo sequencing. The database search method identifies proteins by measuring peptide mass and comparing it to available protein databases. However, this method is unsuitable for analyzing antibodies, peptides, and proteins whose sequence information is either inaccessible or not included in any databases. In these cases, de novo sequencing is the only way to decipher the protein sequence.

 

De novo protein sequencing is a method that does not rely on existing protein sequence databases. Instead, it uses mass spectrometry to analyze protein samples. The principle is based on fragmenting proteins or peptides in a mass spectrometer and interpreting the resulting spectra to determine the amino acid sequence.

 

1. Sample Preparation

In de novo sequencing, protein samples are first digested into smaller peptides using enzymes. Commonly used enzymes include trypsin, Lys-C, and Glu-C.

 

2. Mass Spectrometry Analysis

High-resolution mass spectrometry (such as Orbitrap Fusion Lumos) is used to measure the peptides. The mass spectrometer ionizes the peptides using electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) and measures their mass-to-charge ratio (m/z) in the mass analyzer.

 

3. Peptide Fragmentation

The ionized peptides are further fragmented in the mass spectrometer, producing a series of fragment ions of different lengths. Common fragmentation methods include collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD).

 

4. Data Analysis

By analyzing the spectra of the fragment ions, algorithms and software tools (such as PEAKS or Byonic) reconstruct the amino acid sequence of the peptides. De novo sequencing involves carefully comparing and matching fragment ion signals to determine the position of each amino acid.

 

Advantages and Disadvantages of De Novo Protein Sequencing

1. Advantages

(1) Database Independence: It does not depend on existing databases, making it suitable for analyzing any unknown protein sequences.

(2) High Accuracy: It can accurately identify antibody complementarity-determining regions (CDRs) and other important domains.

(3) Modification Analysis: It is suitable for analyzing various types of protein modifications, such as phosphorylation, acetylation, and glycosylation.

 

2. Disadvantages

(1) Complex Data Analysis: The data analysis process is complex and time-consuming, requiring high-performance computing and specialized software.

(2) High-Quality Data Requirement: High-quality mass spectrometry data is required, necessitating rigorous sample preparation and mass spectrometry operation.

 

Applications in Biomedical Research

1. Antibody and Biomarker Discovery

Identifying newly discovered antibodies and biomarkers.

 

2. Drug Target Analysis

Analyzing drug targets and mechanisms of action.

 

3.Protein Modification Studies

Studying complex protein modifications and their roles in diseases.

 

By leveraging high-resolution mass spectrometry and advanced bioinformatics tools, this technology provides high-quality protein sequence information, supporting biological research and biopharmaceutical development. MtoZ Biolabs provides integrate De Novo protein sequencing service.