Principle of Cellular Proteomics

Cellular proteomics is a crucial field focused on the composition, structure, function, and interactions of proteins within cells. 

 

Protein Extraction and Separation

The first step is the extraction of proteins from cells. During this process, lysis buffers are typically employed to disrupt cell membranes and release intracellular proteins. The composition of lysis buffers often includes detergents, salts, buffers, and other additives to ensure the stability and activity of proteins.

 

After protein extraction, the extracted proteins need to be separated to remove interfering substances and obtain pure protein samples. Common separation techniques include gel electrophoresis and liquid chromatography. Gel electrophoresis can separate proteins based on their size and charge, while liquid chromatography can separate proteins based on their polarity. These techniques not only achieve high-resolution separation but also provide high-quality samples for subsequent mass spectrometric analysis.

 

Protein Identification

Mass spectrometry is the most commonly used method for protein identification from purified protein samples. It can determine the molecular weight of proteins and infer the sequence information by analyzing peptide fragments. Typically, proteins are first digested with enzymes (such as trypsin) to break them down into smaller peptide fragments. Subsequently, mass spectrometry is employed to detect the mass-to-charge ratio (m/z) data of the peptide fragments. By comparing these data with known protein databases (like UniProt and NCBI), rapid and accurate protein identification can be achieved.

 

Protein Quantification

In cellular proteomics research, quantitative analysis is a crucial step in understanding the changes in protein expression under different conditions. Common quantitative methods include labeling quantification (e.g., TMT, iTRAQ) and label-free quantification (e.g., SILAC, Label-free Quantification). Labeling techniques allow for simultaneous comparison of protein expression levels across multiple samples through the addition of specific labels. In contrast, label-free quantification relies on the signal intensity of peptide fragments to analyze the relative abundance between samples. These quantification methods enable researchers to uncover the patterns of protein changes in various physiological or pathological states.

 

Study of Protein Interactions

Proteins within cells often do not exist independently but form complex networks through interactions. Studying protein interactions is essential for understanding cellular functions. Common methods include yeast two-hybrid, co-immunoprecipitation, and pull-down assays. Combined with mass spectrometric analysis, these methods can assist researchers in identifying and characterizing protein complexes and their functions.

 

Cellular proteomics provides us with tools to deeply understand cellular functions and life processes. Through the extraction, separation, identification, and quantitative analysis of proteins, researchers can unveil the complex mechanisms within cells.