The Principles and Processes of Protein De Novo Sequencing Technology

De Novo Sequencing is a technique used to determine the amino acid sequence of proteins or peptides solely based on experimental data, without relying on any known protein or nucleic acid databases. This technology is particularly important in the identification of new proteins or modified proteins.

 

De Novo Sequencing usually utilizes Mass Spectrometry (MS). Proteins or peptides are ionized in the mass spectrometer to produce positive ions. These ions collide with collision gases (such as argon) in the collision cell to generate a series of fragment ions from peptides or proteins. These fragment ions' spectra are known as Tandem Mass Spectra (MS/MS). The fragment ions in MS/MS are formed due to peptide bond cleavage of proteins or peptides, so their mass difference represents the mass of two adjacent amino acid residues.

 

Analysis Workflow

1. Sample Preparation

The purified protein or peptide sample is prepared and introduced into the mass spectrometer through an appropriate method.

 

2. Ionization

Proteins or peptides are ionized into ions using Electrospray Ionization (ESI) or Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) technology.

 

3. Mass Spectrometry Analysis

The m/z value of the protein or peptide parent ions is first measured by the mass spectrometer, and then specific parent ions are selected for further collision to produce fragment ions.

 

4. Tandem Mass Spectrometry Analysis

Parent ions collide with collision gases in the collision cell, causing the protein or peptide to split and form a series of fragment ions. The m/z values of these fragment ions are measured, and the Tandem Mass Spectrum is obtained.

 

5. Data Analysis

Specialized software, such as Mascot or PEAKS, is used to analyze the Tandem Mass Spectrum data. The original protein or peptide amino acid sequence is inferred by comparing the mass differences between fragment ions.

 

6. Verification

Other experimental methods, such as Edman degradation or synthesis of corresponding peptides, are used for verification.

 

The development of De Novo Sequencing technology has largely benefited from advances in mass spectrometry technology. With the improvement of instrument sensitivity and resolution, as well as advancements in data processing software, this technology will be more widely applied in the fields of proteomics and biomedicine.