Image modified from Freepik

Haines et al. present a highly optimised workflow for proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples, addressing critical challenges in mass spectrometry-based proteomics. The researchers out of the Carr lab developed a partially automated, plate-based approach that enables reproducible deep-scale proteomic profiling, achieving approximately 8,000 – 10,000 unique proteins and up to 14,000 localised phosphosites per sample.

The research team used the Covaris Adaptive Focused Acoustics (AFA) sonicator for tissue processing, an Opentrons OT-2 robot for liquid handling, and positive pressure manifold-assisted S-Trap digestion. The workflow eliminates traditional xylene-based deparaffinisation, enhances processing efficiency, and reduces sample loss and contamination.

The method was validated across multiple instrument platforms, including the Exploris 480, timsTOF HT, and Orbitrap Astral, demonstrating high quantitative reproducibility and protein identification depth. Researchers coupled the Astral with the IonOpticks Aurora Elite XT 15×75 C18 UHPLC column, enabling precise peptide separation.

By processing up to 96 samples in less than 4.5 days, the technique significantly accelerates proteomics research, particularly for archived FFPE tissue repositories.

The study could potentially unlock molecular insights from historical tissue collections, providing a robust, high-throughput method for proteome and phosphoproteome analysis that could improve biomarker discovery and disease mechanism understanding.

Publication
bioRxiv

Authors

Moe Haines, John R. Thorup, Simone Gohsman, Claudia Ctortecka, Chelsea Newton, Dan C. Rohrer, Galen Hostetter, D. R. Mani, Michael A. Gillette, Shankha Satpathy, & Steven A. Carr

Title

High-throughput proteomic and phosphoproteomic analysis of formalin-fixed paraffin-embedded tissue