Parallel Proteomic Workflow for Mass Spectrometric Analysis of Tissue Samples Preserved by Different Methods.

Area of Science:

• Biochemistry
• Proteomics
• Biobanking

Background:

• Formalin-fixed paraffin-embedded (FFPE) and frozen tissues are valuable in biobanks but challenging for proteomic analysis.
• Existing methods for mass spectrometry (MS) studies are limited by protein structure and embedding material contamination.

Purpose of the Study:

• To develop a parallel proteomic workflow for FFPE and frozen tissue samples.
• To enable large-scale, reproducible, qualitative, and quantitative high-resolution MS analysis.
• To assess protein expression and post-translational modifications (PTMs) in clinical samples.

Main Methods:

• Optimized protocol for embedding material removal, protein extraction, and multienzyme digestion using filter-aided sample preparation.
• Parallel analysis of FFPE and OCT/frozen bladder cancer samples from two cohorts.
• Titanium dioxide enrichment for phosphopeptide identification.

Main Results:

• Identification of over 2000 proteins in FFPE and 3000 proteins in OCT/frozen samples.
• Clustering of samples into different tumor stages based on protein expression profiles.
• Detection of over 250 phosphopeptides in FFPE and 400 in OCT/frozen samples.
• MS spectra reviewed for contaminations.

Conclusions:

• The developed workflow is effective for large-scale, high-resolution MS analysis of FFPE and frozen tissues.
• The method allows for reproducible protein and site-specific phosphorylation detection in long-term stored clinical samples.
• The study provides insights into similarities and differences between FFPE and frozen tissue proteomes.