Abstract
<jats:p>Formalin fixed paraffin embedded (FFPE) tissue is the dominant clinical pathology resource yet whether it faithfully preserves organ signalling biology and supports directional regulatory analysis remains unquantified. We generated a phosphoproteome map from eight healthy rat organs, separating preservation effects from biological variation. Using mass spectrometry, we quantified 54,710 phosphosites on 5,994 proteins across receptors, kinase cascades and nuclear regulators. Organ specific phosphosite signatures matched known physiological and proliferative states. Paired antagonistic phosphosites converted into "activating minus inhibitory" indices that quantified net tissue specific pathway activity, while a "kinase-by-organ activity" matrix resolved functional hierarchies. Joint analysis with an external fresh frozen phosphoproteome dataset yielded 58,631 phosphosites total, recovering 86% of the 28,888 sites detected in the frozen dataset. Organ identity explained over 92% of the total variance after batch correction, versus under 0.5% for preservation method. Per-organ phosphosite intensities agreed closely between preservation modes except in brain. This establishes that archived pathology tissue supports biologically faithful phosphoproteome analysis at organ, pathway, and site resolution, providing a framework for retrospective signalling studies in clinical archives.</jats:p>