The Basic Processing Procedure of Protein Sequencing Sample

Protein sequencing generally refers to determining the precise order of amino acid residues in a protein molecule, which is a key step in understanding protein function and structure. The main technical means to achieve this is mass spectrometry, especially tandem mass spectrometry (MS/MS). Below is the basic processing procedure for protein sequencing samples.

 

Protein Extraction

1. Cell Lysis

Disrupt the cell membrane to release the intracellular proteins by using physical or chemical methods (such as ultrasonication, freeze-thawing, detergent lysis, etc.).

 

2. Impurity Removal

Remove fats, nucleic acids (DNA/RNA), and other non-protein components. Common methods include centrifugation, filtration, precipitation, etc.

 

Protein Purification

1. Chromatography

Further purify the target protein by using techniques such as ion exchange chromatography, affinity chromatography, gel filtration chromatography, reverse-phase chromatography, etc.

 

2. Protein Concentration

Concentrate the protein solution through ultrafiltration or freeze-drying.

 

Protein Quantification

1. Spectrophotometer

Estimate protein concentration by measuring absorbance (such as A280).

 

2. Colorimetric Method

Like Bradford, BCA, etc., quantify protein through color change.

 

Protein Digestion

1. Enzymatic Hydrolysis

Mix the protein solution with a specific enzyme (usually trypsin), and incubate under suitable temperature and pH conditions, allowing the protein to be enzymatically cleaved into peptide fragments at specific sites.

 

Peptide Purification and Separation

1. Liquid Chromatography

High-performance liquid chromatography (HPLC) is commonly used to separate the digested peptide mixture.

 

2. Clean-Up

Use C18 columns or other solid-phase extraction materials to remove salts and other impurities.

 

Protein Quantitation

1. Labeling Quantitation

Methods such as isotope labeling, label-free quantitation can be performed before or after the digestion step.

 

2. Non-Labeling Quantitation

Such as peak area-based quantitation, etc.

 

Sample Preparation for Mass Spectrometry

1. Ionization

Convert the peptide sample into charged particles through electrospray or MALDI technology.

 

2. Sample Loading

Load the processed sample onto the mass spectrometer.

 

Mass Spectrometry Analysis

1. MS Analysis

Obtain the mass-to-charge ratio of peptides.

 

2. MS/MS Analysis

Fragment the selected peptide ions to obtain the mass spectrum of fragment ions, which is used to determine the amino acid sequence.

 

Data Processing and Analysis

1. Peptide Spectrum Matching

Compare experimental data with theoretical data to identify peptide sequences.

 

2. Bioinformatics Analysis

Further analyze protein function, post-translational modifications, etc.

 

Results Verification and Interpretation

1. Results Confirmation

Verify the accuracy of experimental results.

 

2. Biological Significance Analysis

Interpret the function and biological significance of proteins.

 

The entire process requires the operator to have a certain technical background and also demands high requirements for various instruments and experimental materials. With the development of technology, automated and high-throughput protein sequencing techniques are gradually becoming popular.