High-Throughput Proteomic and Phosphoproteomic Analysis of Formalin-Fixed Paraffin-Embedded Tissues.

Formalin-fixed, paraffin-embedded (FFPE) patient tissues are a valuable resource for proteomic studies with the potential to associate derived molecular insights with clinical annotations and outcomes. Here, we present an optimized, partially automated, plate-based workflow for FFPE proteomics combining pathology-guided macrodissection, xylene-free deparaffinization using Adaptive Focused Acoustics sonication for lysis and decrosslinking, optimized suspension trapping digestion and cleanup of peptides, and LC-MS/MS using Exploris 480, Orbitrap Astral, and timsTOF HT instrumentation. The workflow enables analysis of up to 96 dissected FFPE tissue samples or 10 μm scrolls, identifying 8000 to 10,000 unique proteins per sample with median CVs <20%. Application to lung adenocarcinoma FFPE blocks confirms the platform's effectiveness in processing complex, clinically relevant samples, achieving deep proteome coverage and quantitative robustness comparable to tandem mass tagbased methods. Using the Orbitrap Astral with short, 24-min gradients, the workflow identifies up to 10,000 unique proteins and 11,000 fully localized phosphosites in lung adenocarcinoma FFPE tissue, demonstrating the ability to derive biologically relevant phosphoprotein/peptide results from clinically derived FFPE tumor samples. This high-throughput, scalable workflow advances biomarker discovery and proteomic research in archival tissue samples.