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

Abstract

A mass spectrometry-based methodology has been developed to screen for changes in site-specific core-fucosylation (CF) of serum proteins in early stage HCC with different etiologies. The methods involve depletion of high-abundance proteins, trypsin digestion of medium-to-low-abundance proteins into peptides, iTRAQ labeling, and Lens culinaris Agglutinin (LCA) enrichment of CF peptides, followed by endoglycosidase F3 digestion before mass spectrometry analysis. 1300 CF peptides from 613 CF proteins were identified from patients sera, where 20 CF peptides were differentially expressed in alcohol (ALC)-related HCC samples compared with ALC-related cirrhosis samples and 26 CF peptides changed in hepatitis C virus (HCV)-related HCC samples compared with HCV-related cirrhosis samples. Among these, we found three CF peptides from fibronectin upregulated in ALC-related HCC samples compared with ALC-related cirrhosis samples with an AUC (area under the curve) value of 0.89 at site 1007 with a specificity of 85.7% at a sensitivity of 92.9% (generated with 10-fold cross-validation). When combined with the AFP index, the AUC value reached to 0.92 with a specificity of 92.9% at a sensitivity of 100%, significantly improved compared to that with AFP alone (LR test p < 0.001). In HCV-related samples, the CF level of cadherin-5 at site 61 showed the best AUC value of 0.75 but was not as promising as that of fibronectin site 1007 for ALC-related samples. The CF peptides of fibronectin may serve as potential biomarkers for early stage HCC screening in ALC-related cirrhosis patients.

Single Amino Acid Variant Profiles of Subpopulations in the MCF-7 Breast Cancer Cell Line

“…The details of the gradient are as in our previous work. 30 The mass spectra data acquisition was performed using the Xcalibur software (Thermo Fisher Scientific, CA) in data-dependent mode. The ESI spray voltage was set at 2.5 kV.…”

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.…”

Biomarkers for Hepatocellular Carcinoma

“…Recently, there has been noticeable progress in applying proteomics to the identification of potential HCC biomarkers. Yin et al screened the sera of patients with different liver diseases for core-fucosylated (CF) proteins using a mass spectroscopic approach, which revealed 3 CF peptides from fibronectin at site 1007 with a sensitivity score of 85.7% and specificity of 92.9% for discriminating HCC from cirrhosis in patients with alcohol liver diseases 93. Moreover, they found CF cadherin-5 at site 61 to be the best candidate for distinguishing between HCV-linked HCC tissues and their cirrhosis counterparts, albeit with less effectiveness.…”