Workflow of De Novo Peptide Sequencing

De Novo peptide sequencing is a technique used to determine the primary structure of proteins without relying on known sequences in a database. This method is particularly important for studying newly discovered proteins or organisms that have not been fully investigated. By analyzing mass spectrometry data, researchers can deduce the amino acid sequence of peptide fragments.

 

Workflow

1. Sample Preparation

Sample preparation is the first step in De Novo peptide sequencing, and obtaining high-quality protein samples is crucial. Typically, sample preparation involves the following steps:

 

(1) Protein Extraction

Extract proteins from biological samples, ensuring that the purity and concentration of the samples are sufficiently high.

 

(2) Protein Digestion

Use enzymes such as trypsin to break down proteins into smaller peptide fragments, making them amenable to subsequent mass spectrometry analysis.

 

(3) Peptide Purification

Separate and purify peptide fragments using liquid chromatography techniques to remove impurities.

 

2. Mass Spectrometry Analysis

Mass spectrometry analysis is the core step of De Novo peptide sequencing, where peptide mass spectra are obtained using a mass spectrometer. This process includes:

 

(1) Electrospray Ionization (ESI) or Matrix-Assisted Laser Desorption/Ionization (MALDI)

Ionize the peptide fragments so that they can be detected by the mass spectrometer.

 

(2) Mass Spectrometer Detection

The mass spectrometer detects ions based on their mass-to-charge ratio (m/z) and generates mass spectra. Tandem mass spectrometry (MS/MS) is commonly used to provide more detailed peptide information.

 

(3) Data Acquisition

Collect mass spectra data on a computer for subsequent analysis.

 

3. Mass Spectrometry Data Processing

Data processing is a critical step in translating mass spectra into peptide sequences. This process usually involves the following steps:

 

(1) Spectrum Preprocessing

Remove background noise, smooth spectra, and improve the signal-to-noise ratio.

 

(2) Peak Identification

Identify peaks in the mass spectra, determining the m/z value and intensity of each peak.

 

(3) Peptide Fragment Matching

Deduce the amino acid sequence of peptide fragments by matching theoretical peptide fragments with the peaks in the actual mass spectra.

 

4. Sequence Assembly

Once partial sequences of peptide fragments are obtained, these sequences need to be assembled into complete peptide sequences. This step typically includes:

 

(1) Overlap Sequence Assembly

Assemble longer sequences by overlapping regions of peptide fragments.

 

(2) Sequence Verification

Ensure the accuracy of the assembled sequences by comparing them with known sequences or conducting further experimental verification.

 

5. Result Analysis and Interpretation

The final step is to analyze and interpret the obtained peptide sequences. This may include:

 

(1) Function Prediction

Predict the potential biological functions based on the peptide sequences.

 

(2) Structural Analysis

Further study the protein structure using other techniques such as X-ray crystallography or nuclear magnetic resonance.

 

(3) Database Comparison

Compare the De Novo sequenced peptides with known sequences in databases to find similar sequences and infer their possible origin or function.

 

De Novo peptide sequencing is a powerful technique that allows researchers to accurately determine the amino acid sequence of proteins without reference sequences. Through steps such as sample preparation, mass spectrometry analysis, data processing, sequence assembly, and result analysis, researchers can deduce peptide sequences from mass spectrometry data, providing valuable information for protein function studies.