Mass-Selected Site-Specific Core-Fucosylation of Ceruloplasmin in Alcohol-Related Hepatocellular Carcinoma

Abstract

A mass spectrometry-based methodology has been developed to study changes in core-fucosylation of serum ceruloplasmin that are site-specific between cirrhosis and hepatocellular carcinoma (HCC). The serum samples studied for these changes were from patients affected by cirrhosis or HCC with different etiologies, including alcohol, hepatitis B virus, or hepatitis C virus. The methods involved trypsin digestion of ceruloplasmin into peptides followed by Endo F3 digestion, which removed most of the glycan structure while retaining the innermost N-acetylglucosamine (GlcNAc) and/or core-fucose bound to the peptide. This procedure simplified the structures for further analysis by mass spectrometry, where four core-fucosylated sites (sites 138, 358, 397, and 762) were detected in ceruloplasmin. The core-fucosylation ratio of three of these sites increased significantly in alcohol-related HCC samples (sample size = 24) compared to that in alcohol-related cirrhosis samples (sample size = 18), with the highest AUC value of 0.838 at site 138. When combining the core-fucosylation ratio of site 138 in ceruloplasmin and the alpha-fetoprotein (AFP) value, the AUC value increased to 0.954 (ORsite138 = 12.26, p = 0.017; ORAFP = 3.64, p = 0.022), which was markedly improved compared to that of AFP (AUC = 0.867) (LR test p = 0.0002) alone. However, in HBV- or HCV-related liver diseases, no significant site-specific change in core-fucosylation of ceruloplasmin was observed between HCC and cirrhosis.

Mass-Selected Site-Specific Core-Fucosylation of Serum Proteins in Hepatocellular Carcinoma

“…18 In this study, we screened site-specific CF changes of serum glycoproteins in liver cirrhosis and HCC patients with two etiologies, including ALC and HCV, in order to search for additional specific changes that can be used as markers of HCC. The workflow is shown in Figure S1.…”

Site-Specific Fucosylation Analysis Identifying Glycoproteins Associated with Aggressive Prostate Cancer Cell Lines Using Tandem Affinity Enrichments of Intact Glycopeptides Followed by Mass Spectrometry

“…There are two major strategies for Fuc glycoproteins analyses: (1) targeted Fuc proteins measurement by ELISA, western/lectin blot, or mass spectrometry (MS) 6,13,14,16,19,20 and (2) large-scale Fuc glycoproteomes profiling by MS analysis. 8,10,18,21–24 The second approach may be more comprehensive as it facilitates the understanding of underlying mechanisms associated with tumorigenesis and metastasis as well as the discovery of novel potential biomarkers for early clinical diagnosis.…”

“…Currently, large-scale Fuc glyco-proteomics analyses often employ a divide-and-conquer approach that profiles glycans or deglycosylated peptides separately, utilizing enzymatic or chemical methods to selectively cleave either the glycan or glycopeptide. 8,10,18,20–25 However, a key drawback of this strategy is that information linking the site of glycosylation to its respective glycan structure is lost, and aspects such as glycan microheterogeneity at specific glycosites cannot be examined. 26 Therefore, strategies to profile the large-scale glycoproteomes, including Fuc, by analyzing intact glycopeptides directly are needed.…”

Glycans and glycoproteins as specific biomarkers for cancer

“…Mass spectrometry is a versatile technique that has increasingly been used in cancer biomarkers discovery due to its sensitivity, potential for high throughput, and the capability for tolerating diverse biological mixtures [21–27]. MS has been used in the analysis of various analytes in many fields including but not limited to proteomics, metabolomics, lipidomics, glycoproteomics, glycomics and proteoglycomics [28–31].…”