Time‐Enduring Proteomic Fidelity in Over 30‐Year‐Old FFPE Tissues: Distinct Proteomic Signatures of Hepatocellular Carcinoma and Adjacent Non‐Tumor Liver Tissue

Formalin-fixed paraffin-embedded (FFPE) tissues are crucial clinical archives linked with long-term follow-up data, yet the suitability of deep proteomic analysis on samples stored over 30 years remains largely unexplored. This study aimed to verify the feasibility of deep proteomic analysis on extremely long-term stored FFPE samples. We employed adaptive focused acoustics (AFA) technology for efficient protein extraction, combined with SP3 cleanup and data-independent acquisition (DIA) mass spectrometry using a ZenoTOF 7600, to analyze FFPE samples of hepatocellular carcinoma (HCC) and adjacent non-tumor liver (NTL) tissues from 19 HCC patients from 1988 to 1992. Our workflow identified and quantified approximately 7000 proteins, with excellent reproducibility. Proteomic profiles clearly distinguished HCC from NTL tissues. We identified 630 differentially expressed proteins (467 upregulated in HCC, 163 upregulated in NTL). Pathway analysis revealed expected biological differences: HCC showed enrichment in proliferation/genomic maintenance pathways (e.g., ribosome biogenesis, DNA replication), while NTL showed enrichment in metabolic pathways (e.g., cytochrome P450), consistent with known biology and validated by COSMIC database. Comprehensive, biologically relevant proteomic data can be obtained from FFPE archives over 30 years old. Our validated workflow unlocks the potential of these historically invaluable specimens for powerful retrospective studies, contributing to our understanding of cancer such as HCC.