Proteomic Trajectories of Metabolic and Proteostatic Adaptation During Normothermic Liver Perfusion

<h4>Background</h4>Normothermic machine perfusion (NMP) enables metabolic restoration and viability testing of liver grafts, but current viability criteria incompletely predict post-transplant outcomes. The molecular basis of graft resilience or biliary vulnerability remains unclear. This study aimed to characterise tissue-level proteomic trajectories during NMP and early reperfusion to identify molecular signatures associated with biliary complications after liver transplantation (LT).<h4>Methods</h4>This prospective, single-centre study was conducted at Rennes University Hospital. Twenty donation-after-brain-death (DBD) livers underwent NMP; sixteen transplanted grafts with complete sequential biopsies and ≥ 6 months of follow-up were analysed. Biopsies were collected after cold storage (B1), at the end of NMP (B2), and 1 h after graft reperfusion (B3). Proteins were quantified by high-resolution LC-MS/MS and analysed with Proteome Discoverer 3.1/Chimerys. Pathway enrichment used Ingenuity Pathway Analysis to compare grafts with and without biliary complications.<h4>Results</h4>Principal component analysis revealed distinct proteomic profiles between grafts with and without complications at all biopsy time points. During NMP (B2/B1), uncomplicated grafts showed glycolytic activation with attenuation of oxidative phosphorylation, whereas complicated grafts showed blunted glycolysis and mild OXPHOS upregulation. At reperfusion (B3/B2), complicated grafts displayed induction of translational and endoplasmic-reticulum-stress pathways, while resilient grafts maintained proteasome-related protein turnover and enrichment of a hypoxia-response signature driven by ELOC and proteasome subunits.<h4>Conclusions</h4>Sequential tissue proteomics during NMP reveals divergent metabolic and proteostatic adaptations linked to biliary outcomes. Glycolytic activation with preserved protein turnover characterises resilient grafts, whereas translational and ER-stress programmes predominate in complicated ones. These insights may refine viability assessment beyond biochemical criteria.