How to Use Proteomics to Screen Differential Proteins

Proteomics, as an important part of systems biology, aims to comprehensively analyze the expression, function, and interaction of all proteins in biological organisms. With the continuous deepening of biomedical research, proteomics is playing an increasingly important role in the research of disease mechanisms, the discovery of biomarkers, and the development of new drugs. Particularly in the screening of differential proteins, proteomics technology provides a powerful tool, helping researchers reveal the dynamic changes of disease-related proteins, thereby promoting the understanding of the nature of diseases and the innovation of treatment methods. This process involves multiple steps, from sample preparation to data analysis, each of which is critically important.

 

Analysis Workflow

1. Sample Preparation and Processing

The first step in the screening of differential proteins is the preparation and processing of samples. In this stage, researchers need to collect relevant biological samples, such as diseased and normal tissues, cells or body fluids in disease and recovery states, and through protein extraction, purification, and quantification methods, obtain proteins to be analyzed.

 

2. Protein Separation and Identification

(1) Traditional two-dimensional gel electrophoresis technology and modern liquid chromatography technology (HPLC) are commonly used separation methods. Through these techniques, complex protein samples can be separated into single proteins or peptides, providing a basis for subsequent identification and analysis.

(2) Protein identification usually relies on mass spectrometry technology, which can provide accurate mass and sequence information for proteins or peptides. Data from mass spectrometry analysis can accurately identify the types and expression levels of proteins in the sample by comparing it with databases.

 

3. Protein Quantification

(1) Labeling quantification: Techniques such as isotope-coded relative and absolute quantification (iTRAQ), stable isotope-labeled amino acids (SILAC), etc.

(2) Label-free quantification: Depends on the ion intensity of peptides for protein quantification.

 

4. Data Analysis and Differential Protein Screening

The large amounts of data generated by high-throughput protein analysis need to be processed and analyzed with professional software. These software can identify the identity of proteins, quantify their expression levels, and through statistical analysis (such as t-test, ANOVA), screen for significantly differentially expressed proteins. Differentially expressed proteins can be screened by setting a threshold (such as the fold change in expression level and statistical significance).

 

5. Function Analysis and Verification

Differentially expressed proteins screened out need to be further analyzed by bioinformatics to explore their roles and functions in biological processes. In addition, techniques such as Western blot, enzyme-linked immunosorbent assay (ELISA), or immunofluorescence can be used to independently verify the differentially expressed proteins screened out.

 

Precautions

1. Ensure the repeatability and consistency of sample processing, protein extraction, and digestion processes.

2. The quality of mass spectrometry data directly affects the accuracy of protein identification and quantification.

3. Statistical analysis should consider sample size, the complexity of experimental design, and data variability.