Glycoproteomic Analysis of Urinary Extracellular Vesicles for Biomarkers of Hepatocellular Carcinoma

Abstract

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.

Spotlights on extracellular vesicles in hepatocellular carcinoma diagnosis and treatment: an update review

“…Furthermore, it was shown that the abnormal glycosylation of EV membrane proteins holds potential as an effective noninvasive biomarker for HCC diagnosis ( Li et al, 2023 ). Lin J et al isolated EVs from HCC and adjacent liver tissues for miRNA-level sequencing.…”

Comprehensive Overview the Role of Glycosylation of Extracellular Vesicles in Cancers

“…These findings suggest that profiling serum exosomal N -glycome may yield novel insights into potential HCC biomarkers. Li et al identified 756 N -glycopeptides in urinary EVs and found that glycoproteins LG3BP, PIGR, and KNG1 are upregulated in HCC128 . Although this study had a small sample size and the candidate markers were not validated, this study highlights the promise of site-specific glycomics analysis in the quest for new noninvasive diagnostic markers.…”

Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP–MS

“…In addition, sEVs also show great potential in medical applications. In addition to playing increasingly significant roles in disease biomarker screening, − sEVs are also believed to serve as a novel drug delivery system with intrinsic abilities to pass the blood–brain barrier and exhibit excellent biocompatibility and stability . Extensive research has focused on how sEVs interact with their recipient cells, i.e., recognition by cell surface receptors for signal transduction without cargo delivery into recipient cells, release of contents by means of vesicle-cell membrane fusion, , and a more general way to pass through the cell membrane and travel to a final destination .…”