Proteomics Workflow in Paraffin-Embedded Tissue Samples

Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples are frequently used in clinical pathology for long-term preservation of biological specimens. The formalin fixation process, while preserving tissue structure, induces protein cross-linking, presenting challenges for downstream proteomics analysis. However, with carefully optimized workflows, researchers can successfully perform proteomic studies on FFPE tissues.

 

Deparaffinization and Rehydration

The initial step involves deparaffinizing FFPE samples, typically using xylene, followed by a series of ethanol washes to rehydrate the tissue back into an aqueous environment. This critical process ensures the removal of paraffin, thus preparing the sample for effective protein extraction and subsequent steps.

 

Protein Extraction and Lysis

After rehydration, proteins are extracted from the tissue using strong lysis buffers, often combined with heat to break the formalin-induced cross-links. Lysis buffers typically include components like urea and beta-mercaptoethanol, which denature proteins and facilitate solubilization. The application of microwave or thermal energy can enhance protein yield from the fixed tissues.

 

Reversal of Cross-Linking

To improve protein digestibility, formalin cross-links must be reversed. This is usually accomplished by heating the samples in an alkaline buffer such as Tris-HCl at pH 8.0, restoring the protein structure and enabling efficient downstream digestion.

 

Protein Digestion

Proteins are then digested into smaller peptide fragments using trypsin. Trypsin cleaves the proteins at specific sites, producing peptides suitable for mass spectrometry analysis. Given the cross-linking modifications in FFPE samples, this digestion step may require extended incubation times or increased enzyme concentrations to ensure complete protein degradation.

 

Peptide Purification

The resulting peptides are purified to remove contaminants using techniques like solid-phase extraction (SPE) or spin column purification. This step is essential to maximize the sensitivity and accuracy of the subsequent mass spectrometry analysis by eliminating potential interferences.

 

LC-MS/MS Analysis

Purified peptides are separated using liquid chromatography (LC) and then analyzed by tandem mass spectrometry (MS/MS). LC separates peptides based on their properties, while MS/MS identifies them based on their mass-to-charge ratio (m/z), providing critical information on peptide sequences. For FFPE-derived samples, high-resolution mass spectrometers are preferred to handle the complexity of the peptide mixtures.

 

Data Analysis

The mass spectrometry data are processed through bioinformatics tools to identify peptides and quantify proteins by matching the mass spectra to protein databases. The analysis accounts for the presence of cross-links, and appropriate adjustments are made during data interpretation to ensure accuracy.

 

Validation and Functional Studies

The final step involves validating the results through replicate experiments and functional assays. Beyond profiling protein expression, FFPE proteomics studies also include functional enrichment analyses and pathway explorations, shedding light on the biological significance of the identified proteins in disease mechanisms.