Integrated Proteome and Phosphoproteome Profiling Reveals Abundance-Independent Phosphorylation Remodeling in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is characterized by extensive molecular heterogeneity, yet changes in protein abundance alone may not fully capture the regulatory alterations that drive tumor biology. Here, we performed integrated whole-proteome and phosphoproteome profiling of paired HCC and adjacent non-tumorous liver tissues to distinguish protein-abundance remodeling from phosphorylation changes not explained by comparable parent-protein abundance changes. Label-free nano-LC-MS/MS data were processed using quality control, filtering, missing-value imputation, normalization, and paired statistical testing. Whole-proteome analysis retained 3653 proteins and identified 314 significantly altered proteins, including 79 increased and 235 decreased proteins in HCC. Phosphoproteome analysis retained 1682 phosphopeptides and identified 189 significantly altered phosphopeptides, including 81 increased and 108 decreased events. Integrated analysis showed that 132 significant phosphopeptides matched quantified parent proteins; 112 were classified as phospho-only events, and 20 showed concordant phosphopeptide and parent-protein changes. Reactome enrichment of phospho-only parent proteins highlighted immune/inflammatory processes, metabolic remodeling, ER-stress/protein-folding responses, apoptosis, RHO/RHOBTB GTPase-related regulation, and post-translational phosphorylation. Candidate prioritization identified phosphorylation events for future validation. Overall, this paired multi-layer proteomic analysis demonstrates that HCC tissue exhibits both protein-abundance remodeling and phosphorylation-level rewiring, supporting integrated proteome–phosphoproteome profiling as a strategy to resolve regulatory alterations in cancer tissue.