Rank Normalization Enables Cross Pathway Comparison in Degradation Resolved Proteome Turnover Analysis

Abstract

Protein homeostasis, or proteostasis, is essential for cellular health, and its disruption contributes to cancer, aging, and neurodegenerative disease. Proteostasis is maintained largely by two intracellular degradation systems: the ubiquitin proteasome system (UPS) and the autophagy lysosomal pathway (ALP). Here, we combine pulse stable isotope labeling by amino acids in cell culture (pSILAC) with selective inhibition of the UPS or ALP to quantify pathway-dependent protein turnover and identify substrates that are preferentially cleared through each route. Direct comparison of these perturbations is complicated by their markedly different response distributions. To address the mismatch in dynamic range, we developed a normalization strategy based on within-condition ranks. Protein stabilization values are converted to percentile scores before comparison across pathways, placing both perturbations on a common relative response scale. Across two independent pSILAC experiments, 5,518 proteins were quantified in at least one condition, and 3,814 yielded high confidence half-life measurements in control, proteasome-inhibited, and lysosome-inhibited conditions. Rank normalization resolved pathway sensitivities that were obscured by direct comparison of absolute half-life changes. Importantly, the method reports relative pathway preference rather than absolute pathway contribution, exchanging effect size magnitude for comparability across perturbations with unequal response distributions. Integration with a curated proteostasis network annotation revealed coherent pathway preferences, including coordinated stabilization of proteasome core and regulatory subunits following lysosomal inhibition. This finding provides proteome-wide quantitative evidence for proteaphagy that complements previous targeted observations. Together, these results establish rank normalization as a practical strategy for comparing degradation resolved proteome turnover across perturbations with different dynamic ranges.