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

Area of Science:

• Proteomics
• Biochemistry
• Pathology

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissues are abundant archival resources in pathology.
• Analyzing post-translational modifications like phosphorylation in FFPE tissues is crucial for research.
• Existing methods for FFPE proteomic analysis face challenges in sensitivity and reproducibility.

Purpose of the Study:

• To evaluate the feasibility of a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples.
• To assess the sensitivity and reproducibility of PRM for analyzing protein phosphorylation in FFPE tissues.
• To determine if formalin fixation impacts the relative quantification of phospho-sites.

Main Methods:

• Development and application of two PRM methods: Direct-PRM (without enrichment) and Fe-NTA-PRM (with phosphopeptide enrichment).
• Analysis of tryptic digests from normal brain and glioblastoma multiforme FFPE tissues.
• Targeted quantification of specific phosphorylated peptides from AHNAK, CAMK2D, EIF4B, and EGFR.

Main Results:

• The Fe-NTA-PRM method demonstrated high reproducibility (CV < 14%) for quantifying targeted phosphorylated peptides in normal brain FFPE tissues.
• Formalin fixation did not hinder the relative quantification of phospho-sites and their ratios.
• Successful determination of phosphorylation ratios for four key peptides.

Conclusions:

• Targeted PRM is a sensitive and reproducible method for analyzing protein phosphorylation in FFPE tissues.
• The developed workflow allows for the study of archival FFPE samples for phosphorylation ratio determination.
• This approach expands the utility of FFPE archives for proteomic research.