Steps of De Novo Protein Sequencing Based on Mass Spectrometry

De Novo protein sequencing involves deducing the amino acid sequence of a protein directly from mass spectrometry data without any reference sequence. This process is intricate and meticulous. 

 

Sample Preparation

1. Protein Extraction

Proteins are extracted from cells or tissues, typically using lysis buffers and sonication.

 

2. Protein Purification

The extracted proteins are purified through centrifugation, filtration, and chromatography to remove impurities.

 

3. Protein Digestion

Specific proteases, such as trypsin, degrade the proteins into peptide fragments. This step is known as enzymatic digestion.

 

Mass Spectrometry Analysis

1. Ionization

Peptide fragments are converted into gas-phase ions. Common ionization methods include Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI).

 

2. Mass Analysis

Ionized peptide fragments are separated based on their mass-to-charge ratio (m/z) using mass spectrometers like TOF (Time-of-Flight) and Orbitrap.

 

3. Fragmentation

Peptide ions are fragmented using techniques such as Collision-Induced Dissociation (CID) and Higher-Energy Collisional Dissociation (HCD). The resulting fragment ions provide sequence information.

 

Data Processing and Spectrum Interpretation

1. Peak Detection

Identifying signal peaks in the mass spectrum corresponding to different ions.

 

2. Fragment Ion Matching

Matching observed fragment ions to possible amino acid sequence fragments to deduce the original peptide sequence.

 

3. Sequence Assembly

Assembling the complete amino acid sequence based on the matching results. Common software includes PEAKS and NovoHMM.

 

Result Validation

1. Experimental Validation

Synthesizing the deduced peptide sequence and performing mass spectrometry analysis to verify if it matches the original sample's mass spectrum.

 

2. Biological Validation

Verifying if the deduced protein sequence is reasonable in a biological context, such as checking if it has the expected function and structure.

 

Mechanism Analysis of De Novo Protein Sequencing Based on Mass Spectrometry

De Novo protein sequencing based on mass spectrometry relies on the mass spectrometer's high-precision mass measurement of peptide fragments and their complex fragmentation patterns. ESI and MALDI ionize peptide fragments through different mechanisms. ESI is suitable for liquid chromatography coupled mass spectrometry (LC-MS), while MALDI is often used for mass spectrometry imaging (MSI).

 

In the mass spectrometer, CID causes fragmentation by accelerating ions to collide with inert gas, making it suitable for lower energy fragmentation. HCD, on the other hand, uses higher energy collisions, generating more complex fragment ion spectra. The mass-to-charge ratio (m/z) of these fragment ions provides characteristic information about the peptide sequence. Decoding this information through sophisticated algorithms allows the assembly of the complete amino acid sequence.

 

By continuously optimizing sample preparation, mass spectrometry analysis, and data processing methods, we can more accurately determine the amino acid sequence of proteins, providing a powerful tool for proteomics research. MtoZ Biolabs provides integrate De Novo protein sequencing service.