Decade‐Resolved Proteomic Profiling of Gastric Cancer FFPE Archives: Evaluating Storage‐Associated Shifts and Signal Stability Over 50 Years

Archival formalin-fixed, paraffin-embedded (FFPE) tissues are invaluable for retrospective clinical research, yet the impact of multi-decade storage on quantitative proteomic fidelity remains poorly understood. We conducted a systematic, decade-resolved proteomic assessment using data-independent acquisition (DIA) LC-MS/MS to evaluate FFPE specimens spanning 50 years. This study included 33 gastric cancer cases collected at approximately 10-year intervals from 1972 to 2022. From each case, tumor tissue, matched non-tumorous mucosa, and muscle layer were macrodissected and processed simultaneously using a standardized workflow to minimize technical bias. Longitudinal analysis across five decades demonstrated that a substantial portion of the detectable proteome remains quantitatively stable. While storage- and archival-era-associated factors contributed to measurable variance, tissue-specific biological signatures were the dominant drivers of proteomic profiles. The muscle layer exhibited the highest stability, serving as a structural reference for evaluating archival ageing. Differential expression analysis between tumor and nontumorous mucosa identified robust pathological signatures that persisted even in 50-year-old specimens. Although we observed era-specific variations in protein detectability, a core set of differentially expressed proteins and their associated functional pathways-including cell cycle and metabolic remodeling-were consistently preserved across the evaluable year groups. Notably, the treatment-naïve 1972 cohort provided high-depth biological insights comparable to contemporary samples. Our findings establish that long-term archival storage does not preclude biologically interpretable proteomic profiles, although storage- and archival-era-associated shifts should be explicitly accounted for in analysis and interpretation.