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.
Highlights
Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is the fifth malignant cancer in the world [1]
In order to explore the heterogeneity of glycosylation in hepatocellular carcinoma (HCC) tissues with different levels of alpha-fetoprotein (AFP) in patient serum, an integrated glycoproteomics and proteomics analysis was performed on two paired groups of HCC-related tissues: ten paired tumor and paracancer tissues with low AFP concentration (< 20 ng/mL) in patients’ sera, and ten paired tumor and paracancer tissues with high AFP concentration [> 1,000 ng/mL, an exclusion criterion in Milan Criteria for liver transplantation in patients with HCC [18]] (Figure 1 and Table S1)
Four Tandem Mass Tag (TMT)-labeled peptide samples were mixed and separated into two aliquots: one aliquot was used for intact glycopeptides enrichment using mixed anion-exchange (MAX) cartridges for site-specific glycosylation analysis, and the other aliquot was directly analyzed by mass spectrometry for global proteomic analysis
Summary
Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is the fifth malignant cancer in the world [1]. By using the above workflow, 4,741 unique N-linked intact glycopeptides were identified from TMT-labeled HCC samples as well as their paracancer tissues within 1% false discovery rate (FDR) (Table S2), which were comprised of 221 Nglycan compositions and 1,184 glycosylation sites from 894 glycoproteins (Figure 2A).
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