Systematic Evaluation of the Impact of Storage Time on Label-Free Proteomics of Colorectal Adenocarcinoma Formalin-Fixed Paraffin-Embedded Tissues

Mass spectrometry (MS)-based proteomics
has empowered comprehensive
protein profiling of biological specimens. However, formalin-fixed
paraffin-embedded (FFPE) tissuescritical resources for clinical
biomarker discovery-remain underexplored in the setting of long-term
storage (>15 years). Herein, we systematically evaluated the impact
of storage time on proteomic analyses of 80 colorectal adenocarcinoma
(CRC) FFPE samples, which were stratified by two key variables: storage
time (>15 years vs <1 year) and tissue type (tumor vs adjacent
normal tissue). We adopted a standardized protein extraction strategy,
and subsequent proteomic profiling was performed via data-dependent
acquisition and data-independent acquisition MS workflows. Our results
demonstrated that FFPE tissue storage time impacts protein extraction
efficiency, peptide yields, PTM identification, and protein quantification.
The impacts were more pronounced on the peptide level. However, the
biological enrichments (Gene Ontology and Kyoto Encyclopedia of Genes
and Genomes enrichment analysis) from the global proteome profile
and from differentially expressed proteins in CRC tissues were independent
of archival time. Five clinically relevant biomarkers of CRC were
further validated via immunohistochemistry. Collectively, our findings
confirm that FFPE tissues retain stability for proteomic analyses
even following >15 years of storage, thereby providing critical
insights
for leveraging archival FFPE biobanks to advance clinical proteomics
and archival pathology research.