Mechanism of Proteomic Analysis in FFPE Samples

Formalin-fixed, paraffin-embedded (FFPE) samples are among the most commonly used specimen types in modern pathology. This process preserves cellular morphology and structure by fixing biological samples in formalin and subsequently embedding them in paraffin. The advantages of FFPE samples include long-term storage and reversibility, offering significant application potential in cancer research, genetic studies, and drug development.

 

Proteomics is the study of all proteins in a cell, tissue, or organism, involving identification, quantification, and functional analysis. By analyzing the proteome, researchers can gain insights into physiological states, disease mechanisms, and treatment responses.

 

Processing and Analysis Mechanisms of FFPE Samples

1. Protein Extraction

Extracting proteins from FFPE samples is the first step in proteomic analysis. Since formalin fixation can lead to protein cross-linking and denaturation, the extraction process often employs strong chemical agents, such as urea or thiourea, to break these links and restore the proteins' original structure. Typical steps include deparaffinization, removal of formalin residues, and cell lysis through ultrasonic or mechanical means.

 

2. Protein Digestion

After extraction, proteins must be digested, typically using trypsin. This process breaks proteins into smaller peptide segments for subsequent mass spectrometry analysis. The digestion time and enzyme concentration need to be strictly controlled to ensure uniformity and efficiency.

 

3. Protein Labeling and Separation

Prior to mass spectrometry analysis, researchers often label peptide segments to enhance sensitivity and specificity. Common labeling methods include TMT (Tandem Mass Tag) and iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). Following this, liquid chromatography (LC) is used to separate the labeled peptides to obtain clear signals during mass spectrometry analysis.

 

4. Mass Spectrometry Analysis

Mass spectrometry (MS) is the core technology for proteomic analysis of FFPE samples. Through mass spectrometry, researchers can identify and quantify digested peptide segments. Commonly used techniques include LC-MS/MS (liquid chromatography-tandem mass spectrometry), which, with its superior resolution and sensitivity, has become the gold standard in current proteomic analysis.

 

5. Data Analysis and Interpretation

The data obtained from mass spectrometry undergo a complex analytical process, including data preprocessing, peptide identification, protein quantification, and functional annotation. Common software tools include MaxQuant and Proteome Discoverer, which assist researchers in extracting useful information and conducting bioinformatics analyses.