Phosphorylation ratio determination in fresh frozen and formalin-fixed paraffin embedded tissue with targeted mass spectrometry

ABSTRACT: Formalin-fixed paraffin embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. Therefore, FFPE tissues are 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. Normal brain and glioblastoma multiforme (GBM) tissues were used to demonstrate the feasibility of our approach. Two analytical methods (or workflows) were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). Applying these two methods, the phosphorylation ratio could be determined for selected four 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, using the Fe-NTA-PRM method, we were able to quantify the targeted phosphorylated peptides with a high degree of reproducibility (CV = 14%). Our results indicate that formalin fixation does not impede relative quantification of a phosphosite and its phosphorylation ratio in FFPE tissues. The developed methods open ways to study archival FFPE tissues.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

DISEASE(S): Glioblastoma

SUBMITTER: Lona Zeneyedpour  

LAB HEAD: Theo Luider

PROVIDER: PXD017943 | Pride | 2020-08-25

REPOSITORIES: Pride