Workflow of De Novo Protein Sequencing

Proteins are the main functional molecules within organisms, and their amino acid sequences determine their three-dimensional structures and biological functions. Therefore, deciphering the amino acid sequences of proteins is crucial for understanding their functions.

 

Sample Preparation

1. Protein Extraction

Extract proteins from cells or tissues, ensuring the protein samples are pure and free from contamination.

 

2. Protein Digestion

Use specific enzymes (such as trypsin) to break down the proteins into smaller peptides for subsequent mass spectrometry analysis.

 

Mass Spectrometry Analysis

1. Peptide Ionization

Convert peptides into gas-phase ions using electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI).

 

2. Mass-to-Charge Ratio Measurement

Measure the mass-to-charge ratio (m/z) of the peptide ions in the mass spectrometer to obtain a series of precursor ion spectra.

 

3. Peptide Fragmentation

Further fragment the precursor ions using methods such as collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), or electron transfer dissociation (ETD) to generate fragment ion spectra.

 

Data Analysis

1. Spectra Interpretation

Use specialized software (such as PEAKS or Byonic) to interpret the mass spectrometry data and identify the signals of precursor and fragment ions.

 

2. Sequence Reconstruction

Deduce the amino acid sequences of the peptides based on the fragment ion spectra using algorithms and gradually piece together the complete protein sequence.

 

3. Result Validation

Verify the accuracy of the sequencing results using methods such as reverse sequence confirmation.

 

Result Reporting

1. Experimental Procedures

Describe the detailed steps of sample preparation, mass spectrometry analysis, and data interpretation.

 

2. Mass Spectrometry Parameters

List the parameter settings of the liquid chromatography and mass spectrometer.

 

3. Raw Data Files

Provide the raw data files generated from mass spectrometry analysis.

 

4. Peptide Identification Results

Include the mass-to-charge ratio, intensity, and peptide mapping information of the peptides.

 

5. Protein Sequence and Modification Information

Provide the complete protein sequence and any detected post-translational modifications (PTMs).

 

Although the process is complex and demands high data analysis requirements, its application prospects in protein function research are broad. MtoZ Biolabs provides integrate De Novo protein sequencing service.