Proteomics Analysis of Formalin Fixed Paraffin Embedded Tissues in the Investigation of Prostate Cancer

Prostate cancer (PCa) is one of the leading causes of
death in
men worldwide. The molecular features, associated with the onset and
progression of the disease, are under vigorous investigation. Formalin-fixed
paraffin-embedded (FFPE) tissues are valuable resources for large-scale
studies; however, their application in proteomics is limited due to
protein cross-linking. In this study, the adjustment of a protocol
for the proteomic analysis of FFPE tissues was performed which was
followed by a pilot application on FFPE PCa clinical samples to investigate
whether the optimized protocol can provide biologically relevant data
for the investigation of PCa. For the optimization, FFPE mouse tissues
were processed using seven protein extraction protocols including
combinations of homogenization methods (beads, sonication, boiling)
and buffers (SDS based and urea–thiourea based). The proteome
extraction efficacy was then evaluated based on protein identifications
and reproducibility using SDS electrophoresis and high resolution
LC-MS/MS analysis. Comparison between the FFPE and matched fresh frozen
(FF) tissues, using an optimized protocol involving protein extraction
with an SDS-based buffer following beads homogenization and boiling,
showed a substantial overlap in protein identifications with a strong
correlation in relative abundances (rs = 0.819, p < 0.001). Next, FFPE
tissues (3 sections, 15 μm each per sample) from 10 patients
with PCa corresponding to tumor (GS = 6 or GS ≥ 8) and adjacent
benign regions were processed with the optimized protocol. Extracted
proteins were analyzed by GeLC-MS/MS followed by statistical and bioinformatics
analysis. Proteins significantly deregulated between PCa GS ≥
8 and PCa GS = 6 represented extracellular matrix organization, gluconeogenesis,
and phosphorylation pathways. Proteins deregulated between cancerous
and adjacent benign tissues, reflected increased translation, peptide
synthesis, and protein metabolism in the former, which is consistent
with the literature. In conclusion, the results support the relevance
of the proteomic findings in the context of PCa and the reliability
of the optimized protocol for proteomics analysis of FFPE material.