Abstract
Hepatocellular carcinoma (HCC) remains a leading cause of cancer mortality, and current biomarkers such as alpha-fetoprotein (AFP) lack diagnostic accuracy. Here, we report the first comprehensive profiling of the plasma endogenous phosphopeptidome in HCC, cirrhosis, and healthy controls using a digestion-free LC-MS/MS workflow. From 60 plasma samples, 1,365 phosphopeptides corresponding to 549 proteins were identified and quantified. Among these, the statherin-derived peptide DSSEEKFLR demonstrated outstanding discrimination between HCC and cirrhosis (AUC = 0.968), outperforming AFP (AUC = 0.648). Additional peptides, including PPGAPHTEEEGAE (NST1), YEYDELPAKDD (C4A), SLPGESEEMMEEVD (ITIH4), and VSLGSPSGEVSHPRKT (AHSG), also showed high accuracy (AUC > 0.80). Functional enrichment revealed perturbations in acute-phase response, coagulation, lipid metabolism, and LXR/RXR signaling. Collectively, this work defines a novel plasma phosphopeptide signature that reflects disease-specific proteolytic and phosphorylation dynamics, providing a foundation for developing biomarkers for early detection and clinical monitoring of HCC.