Principle of Proteomics in Paraffin Embedded Samples

Paraffin-embedded tissue proteomics is an essential technique for analyzing the protein composition and expression in tissue samples. The core principle involves the processing of biological samples (such as tumor tissues or other pathological tissues) through fixation, dehydration, and embedding steps, facilitating subsequent mass spectrometry analysis. This technique not only preserves the morphological structure of samples but also provides higher resolution and sensitivity in analyses.

 

Sample Processing

The primary step in paraffin embedding is sample fixation. A commonly used fixing solution is 10% neutral buffered formalin (NBF), which aims to maintain the morphology and structure of tissue cells. After fixation, the samples undergo a dehydration process, typically using increasing concentrations of ethanol, culminating in immersion in paraffin. During this process, the water content within the tissue cells is replaced by ethanol, which is ultimately substituted with molten paraffin.

 

Paraffin Embedding

In the paraffin embedding process, samples are placed in preheated paraffin until fully infiltrated, followed by cooling to form solid blocks. This process ensures the stability of intracellular proteins, preventing potential degradation during subsequent analyses. Once paraffin-embedded, the samples can be stored at room temperature for extended periods without compromising their proteomic analysis.

 

Protein Extraction

The extraction of proteins from paraffin-embedded samples is a critical step for subsequent analyses. Initially, paraffin blocks are sliced to achieve the desired thickness of tissue sections. Then, a deparaffinization agent (such as xylene) is used to remove the paraffin, followed by a series of buffer solutions to extract proteins. This process may involve various chemical reagents to ensure complete solubilization and extraction of proteins.

 

Protein Identification and Quantification

The extracted proteins can be analyzed using liquid chromatography-mass spectrometry (LC-MS). In the LC-MS analysis, proteins are first enzymatically digested (e.g., with trypsin) to generate peptides. Subsequently, liquid chromatography separates the peptide segments, which are then quantified and identified through mass spectrometry. Mass spectrometry provides critical data on protein molecular weight, amino acid sequences, and modification information.

 

The applications of paraffin-embedded tissue proteomics are extensive, covering cancer research, pathology, and basic medicine. By comparing analyses of different samples, researchers can unveil disease mechanisms and identify biomarkers, providing new insights for clinical diagnosis and treatment.