Exploring the Feasibility of Formalin-Fixed Paraffin-Embedded Tissue Phosphoproteomics in Cancer Research

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissues, traditionally used for histopathological examination, are increasingly being utilised for molecular analyses, including phosphoproteomics. This thesis investigates the extent and quality of phosphoproteomes extracted from FFPE tissues using liquid chromatography-mass spectrometry (LC-MS) to evaluate their potential in personalised cancer medicine. A high-throughput workflow was developed to quantify a shallow phosphoproteome from 50 μg of rat tryptic peptides. Optimisation studies identified heptane-methanol as the most reproducible deparaffinisation method, while trifluoroethanol was optimal for phosphoproteome extraction. Data-independent acquisition revealed high reproducibility, with Pearson correlation coefficients exceeding 0.95, and biological relevance, as phosphoproteomic profiles successfully clustered rat organs. Comparisons of FFPE and fresh-frozen tissues demonstrated substantial overlap (85-97% for proteomes, 82-98% for phosphoproteomes), confirming the integrity of the FFPE phosphoproteome. To extend these findings to human cancer, FFPE phosphoproteomics was applied to specimens from HPV-positive oropharyngeal squamous cell carcinoma. Mining existing DIA proteomic data using a FragPipe workflow enabled the identification of phosphoproteomes, which were predictive of patient survival outcomes via multivariate Cox regression. Validation using enriched phosphoproteomes showed limited overlap on a Triple TOF MS but significant improvement with the Orbitrap Astral MS, highlighting the importance of advanced MS technologies. These overall findings demonstrate that FFPE-derived phosphoproteomes are reproducible, biologically informative and may be clinically relevant, supporting their integration into biomarker discovery pipelines and personalised cancer medicine workflows.