Glycoproteomic Analysis of Urinary Extracellular Vesicles for Biomarkers of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) accounts for the most common form of primary liver cancer cases and constitutes a major health problem worldwide. The diagnosis of HCC is still challenging due to the low sensitivity and specificity of the serum α-fetoprotein (AFP) diagnostic method. Extracellular vesicles (EVs) are heterogeneous populations of phospholipid bilayer-enclosed vesicles that can be found in many biological fluids, and have great potential as circulating biomarkers for biomarker discovery and disease diagnosis. Protein glycosylation plays crucial roles in many biological processes and aberrant glycosylation is a hallmark of cancer. Herein, we performed a comprehensive glycoproteomic profiling of urinary EVs at the intact N-glycopeptide level to screen potential biomarkers for the diagnosis of HCC. With the control of the spectrum-level false discovery rate ≤1%, 756 intact N-glycopeptides with 154 N-glycosites, 158 peptide backbones, and 107 N-glycoproteins were identified. Out of 756 intact N-glycopeptides, 344 differentially expressed intact N-glycopeptides (DEGPs) were identified, corresponding to 308 upregulated and 36 downregulated N-glycopeptides, respectively. Compared to normal control (NC), the glycoproteins LG3BP, PIGR and KNG1 are upregulated in HCC-derived EVs, while ASPP2 is downregulated. The findings demonstrated that specific site-specific glycoforms in these glycoproteins from urinary EVs could be potential and efficient non-invasive candidate biomarkers for HCC diagnosis.