Workflow of Cellular Proteomics

Cellular proteomics is an important field that studies the composition, function, and interactions of all proteins within cells. By systematically analyzing cellular proteins, scientists can gain insights into biological processes and their roles in diseases.

 

Sample Preparation

Sample preparation is the first step in cellular proteomics, typically involving the selection of appropriate cell lines or tissue samples. The samples should accurately represent the biological state under investigation. Handling of samples should occur at low temperatures to prevent protein degradation. Cells are then lysed mechanically or enzymatically to release intracellular proteins.

 

Protein Extraction

The protein extraction step primarily involves using an appropriate lysis buffer to solubilize the cell membrane, with commonly used buffers containing detergents. The extracted proteins are then subjected to centrifugation to remove cell debris and insoluble materials. The resulting supernatant contains the target proteins.

 

Protein Separation

Protein separation typically employs electrophoresis techniques, with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) being the most common method. This step separates different proteins based on molecular weight. Additionally, two-dimensional electrophoresis can be used to enhance resolution by separating proteins based on their isoelectric points and molecular weights.

 

Protein Identification

Protein identification is achieved through mass spectrometry analysis. Common methods include MALDI-TOF and LC-MS/MS. Mass spectrometry provides information on protein molecular weight and amino acid sequences. By comparing with databases, the identity of proteins can be determined.

 

Protein Quantification

Protein quantification is a crucial step in analyzing the expression levels of proteins within cells. Common quantification methods include labeling and label-free techniques. Labeling methods such as TMT (Tandem Mass Tags) and iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) allow for comparisons between different samples, while label-free methods like SWATH-MS are suitable for quantitative analysis of the entire proteome.

 

Data Analysis

The obtained mass spectrometry data requires complex computational and statistical analysis. Software tools such as MaxQuant and Proteome Discoverer are used to process proteomics data. This analysis includes protein identification, quantification, and functional annotation, typically organized in tabular form for subsequent analysis and visualization.

 

Bioinformatics Analysis

Following data analysis, bioinformatics analysis is a critical phase that interprets protein functions, pathways, and interaction networks to help understand biological significance. This step often involves online databases and software, such as STRING and DAVID, for enrichment analysis and network construction to reveal potential biomarkers or therapeutic targets.

 

Result Validation

Finally, experimental methods are used to validate the results of data analysis. This may include techniques such as Western blotting, ELISA, or immunofluorescence staining to ensure the reliability and reproducibility of the obtained results.

 

The workflow of cellular proteomics is a systematic and complex process, where each step is vital to the final results. By comprehensively analyzing cellular proteins, scientists can gain deeper biological insights that support disease research and drug development.