Single-shot mass spectrometry-based phosphoproteomic analysis of patient-derived formalin-fixed, parrafin-embedded samples reveal tissue specific fingerprints

Routine in molecular tumor boards for patient therapy evaluation mainly relies on formalin-fixed and paraffin-embedded (FFPE) tissues for multiple screening strategies. While the analysis of the proteome from FFPE has been implemented just recently in MTB screenings, the profiling of post-translational modifications like phosphorylation is not applicable from FFPE samples. Previous approaches for this sample type involve TMT-labeling that is not suitable for individualized and independent patient-level profiling. In addition, validation for a robust analysis of the cell state, fate and functionality on phosphoproteome level remains to be demonstrated. By refining current automated phosphoenrichment protocols, we aim to establish a label-free phosphoproteomic workflow for FFPE-derived tissues. After metal-ion affinity chromatography, we analyze the phosphoproteome of a multi-patient, multi-organic non-cancer cohort on a timsTOF HT in data-independent acquisition. In the explorative analysis more than 6000 phosphopeptides are identified from 50 ug of single samples which enables the accurate tissue-specific profiling in accordance to previously published clinical phosphoproteome data sets. By testing for the coverage of relevant oncogenic signaling pathways, we showcase that the transfer of state-of-the art workflows from research to clinical routine adds valuable insights to personalized medicine. In addition, we aim to control for routinely error-prone tissue preparation steps from the surgery to MS and their effect on the phosphoproteomic level in a time-to-fixation experiment allowing us to implement a pipeline for sample quality assessment.