Workflow of Exosome Proteomics

Exosomes are small membrane vesicles secreted by cells, containing a variety of biomolecules such as proteins, lipids, and RNAs. In recent years, the significance of exosomes in intercellular communication, disease diagnosis, and therapy has become increasingly prominent, making exosome proteomics research vital.

 

Exosome Isolation

The isolation of exosomes is the first step in proteomics research, with main methods including ultracentrifugation, filtration, immunocapture, and commercial exosome isolation kits.

 

1. Ultracentrifugation

(1) Cell Culture

Cells are cultured under appropriate conditions.

 

(2) Collection of Culture Medium

The culture medium is collected and cell debris is removed.

 

(3) Centrifugation Process

Multi-step centrifugation (e.g., 300×g, 2,000×g, 10,000×g, and 100,000×g) is performed to gradually remove larger particles and enrich exosomes.

 

2. Filtration

Membrane filters (e.g., 0.22 μm) are used to remove larger particles, further purifying exosomes.

 

3. Immunocapture

Specific antibodies are used to selectively capture exosomes by binding to surface markers.

 

4. Commercial Kits

Various commercial exosome isolation kits are available on the market, which often combine multiple techniques to enhance isolation efficiency.

 

Protein Extraction

1. Cell Lysis Buffer Method

Appropriate lysis buffers (e.g., RIPA buffer) are used to treat exosomes and release internal proteins.

 

2. Chemical Precipitation

Organic solvent methods, such as chloroform-methanol precipitation, are employed to separate proteins from other biomolecules.

 

3. Sonication

Ultrasound treatment is used to disrupt the cell membrane and release proteins.

 

Protein Quantification

1. Bradford Assay

This method is based on the color change caused by dye binding to proteins, measuring optical absorbance for quantification.

 

2. BCA Assay

This method utilizes disulfide bond reduction reactions and is measured using a UV spectrophotometer for protein quantification.

 

Proteomics Analysis

1. Digestion

Proteins are typically digested using trypsin to convert them into peptides.

 

2. Chromatographic Separation

Liquid chromatography (e.g., LC-MS/MS) is used to separate peptides, enhancing analytical sensitivity.

 

3. Mass Spectrometry Analysis

Mass spectrometry (MS) accurately measures the mass and abundance of peptides, providing essential information for subsequent data analysis.

 

Data Analysis

1. Data Processing

Specialized software is used to process mass spectrometry data, eliminating background noise and performing peak identification.

 

2. Protein Identification

Experimental peptide data is matched against known protein databases to determine the source proteins.

 

3. Quantitative Analysis

Protein abundances are compared across different samples using relative or absolute quantification methods.

 

4. Functional Analysis

Based on identified proteins, pathway enrichment and functional annotation are performed to explore their potential roles in biological processes.

 

Research in exosome proteomics offers new perspectives on understanding intercellular communication, disease mechanisms, and potential biomarkers. Through a systematic workflow, researchers can accurately and efficiently analyze proteins within exosomes, advancing knowledge in related fields.