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

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.