Heterogeneities of Site-Specific N-Glycosylation in HCC Tumors With Low and High AFP Concentrations

Abstract

Hepatocellular carcinoma (HCC) is still one of the malignant tumors with high morbidity and mortality in China and worldwide. Although alpha-fetoprotein (AFP) as well as core fucosylated AFP-L3 have been widely used as important biomarkers for HCC diagnosis and evaluation, the AFP level shows a huge variation among HCC patient populations. In addition, the AFP level has also been proved to be associated with pathological grade, progression, and survival of HCC patients. Understanding the intrinsic heterogeneities of HCC associated with AFP levels is essential for the molecular mechanism studies of HCC with different AFP levels as well as for the potential early diagnosis and personalized treatment of HCC with AFP negative. In this study, an integrated N-glycoproteomic and proteomic analysis of low and high AFP levels of HCC tumors was performed to investigate the intrinsic heterogeneities of site-specific glycosylation associated with different AFP levels of HCC. By large-scale profiling and quantifying more than 4,700 intact N-glycopeptides from 20 HCC and 20 paired paracancer samples, we identified many commonly altered site-specific N-glycans from HCC tumors regardless of AFP levels, including decreased modifications by oligo-mannose and sialylated bi-antennary glycans, and increased modifications by bisecting glycans. By relative quantifying the intact N-glycopeptides between low and high AFP tumor groups, the great heterogeneities of site-specific N-glycans between two groups of HCC tumors were also uncovered. We found that several sialylated but not core fucosylated tri-antennary glycans were uniquely increased in low AFP level of HCC tumors, while many core fucosylated bi-antennary or hybrid glycans as well as bisecting glycans were uniquely increased in high AFP tumors. The data provide a valuable resource for future HCC studies regarding the mechanism, heterogeneities and new biomarker discovery.

Towards structure-focused glycoproteomics

“…While the pre-2016 glycoproteomics studies were found to be rather qualitative in their approaches by simply reporting lists of identified glycopeptides as reviewed [ 22 ], our survey of the post-2018 glycoproteomics papers shows that glycopeptide quantification is now commonly applied. Approaches include label-free quantification by XIC or spectral counting [ 50 , 65 ], isotopic dimethyl [ 54 ] or diethyl [ 55 ] labelling as well as TMT [ 52 , 64 ] and DiLeu [ 39 ] labelling, which collectively enable different types of glycopeptide quantitation strategies all regularly used in the field. While glycoproteome mapping studies typically quantify the relative abundance of all glycoforms observed at each glycosylation site to establish a stoichiometric site-specific glycoprofile, comparative glycoproteomics studies often compare the relative quantity of individual glycopeptides or the glycoprofile at each site between conditions.…”

Proteomic Profiling and Artificial Intelligence for Hepatocellular Carcinoma Translational Medicine

“…Large-scale glycoproteome profiling and quantification of more than 4700 intact N -glycopeptides from 20 HCC and 20 paired paracancer samples enabled distinguishing low and high AFP level HCCs. Several sialylated but not core fucosylated tri-antennary glycans have been found to be uniquely increased in HCC with low AFP level, while many core fucosylated bi-antennary or hybrid glycans as well as bisecting glycans were uniquely increased in high AFP level tumors [ 190 ]. Differential quantitation analysis revealed that five N -glycopeptides at sites N184 and N241 of serum haptoglobin bearing a monofucosylated triantennary glycan were significantly elevated during the progression from non-alcoholic steatohepatitis (NASH) and cirrhosis to HCC.…”

An Optimized Integrin α6-Targeted Magnetic Resonance Probe for Molecular Imaging of Hepatocellular Carcinoma in Mice

“…Alpha fetoprotein (AFP) is the most commonly used of serum biomarkers for HCC diagnosis, but the sensitivity and specificity are only 41-65% and 80-90%. 8,9 Traditional imaging modalities that have clinical relevance in HCC diagnosis include ultrasound (US), computed tomography (CT) and magnetic resonance (MR) imaging. 10,11 US has been extensively used as the preferred imaging modality for screening HCC, with a sensitivity ranging from 40% to 80% and a specificity from 82% to 100%.…”