Abstract
Formalin-fixed, paraffin-embedded (FFPE) patient tissues are a valuable resource for proteomic studies with the potential to associate the derived molecular insights with clinical outcomes. Here we present an optimized, partially automated workflow for FFPE proteomics combining pathology-guided macro-dissection with Adaptive Focused Acoustics (AFA) sonication for lysis and decrosslinking, S-Trap digestion to peptides, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using Orbitrap, Astral or timsTOF HT instrumentation. The workflow enables analysis of up to 96 dissected FFPE tissue samples or whole 10 µM scrolls, identifying 8,000-10,000 unique proteins with <20% median CVs. Key optimizations include improved tissue lysis, protein quantification for normalization, and peptide cleanup prior to LC-MS/MS analysis. Application to lung adenocarcinoma (LUAD) FFPE blocks confirms the platform's effectiveness in processing complex samples, achieving deep proteome coverage and quantitative robustness comparable to TMT-based methods. Using the newly released Orbitrap Astral with short, 24-minute gradients, the workflow identifies up to ∼10,000 unique proteins and ∼11,000 localized phosphosites in LUAD FFPE tissue. This high-throughput, scalable workflow advances biomarker discovery and proteomic research in archival tissue samples.
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