Phosphorylation Ratio Determination in Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Tissue with Targeted Mass Spectrometry

Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. These are, therefore, the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. The feasibility of this technical approach was demonstrated for normal brain and glioblastoma multiforme tissues. Two methods were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). With these two methods, the phosphorylation ratio could be determined for four selected peptide pairs that originate from neuroblast differentiation-associated protein (AHNAK S5448-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), eukaryotic translation initiation factor 4B (EIF4B S93-p), and epidermal growth factor receptor (EGFR S1166-p). In normal brain FFPE tissues, the Fe-NTA-PRM method enabled the quantification of targeted phosphorylated peptides with high reproducibility (CV < 14%). Our results indicate that formalin fixation does not impede relative quantification of a phospho-site and its phosphorylation ratio in FFPE tissues. The developed workflow combining these methods opens ways to study archival FFPE tissues for phosphorylation ratio determination in proteins.