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 1988-1992.