Proteomic profiling of FFPE specimens: Discovery of HNRNPA2/B1 and STT3B as biomarkers for determining formalin fixation durations.

Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long periods. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-term AF-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-period NBF-fixed liver and lung tissues, and granular staining in all long-term AF-fixed FFPE tissue types. Similar trends were observed in the long-period fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. SIGNIFICANCE: The quality of formalin-fixed, paraffin-embedded (FFPE) samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids. and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using liquid chromatography/mass spectrometry (LC/MS) and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation time as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin quality for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics.