Nonalcoholic steatohepatitis (NASH)
is rising in prevalence in
the United States and is a growing cause of hepatocellular carcinomas
(HCCs). Site-specific glycan heterogeneity on glycoproteins has been
shown as a potential diagnostic biomarker for HCC. Herein, we have
performed a comprehensive screening of site-specific N-glycopeptides
in serum haptoglobin (Hp), a reporter molecule for aberrant glycosylation
in HCC, to characterize glycopeptide markers for NASH-related HCCs.
In total, 70 NASH patients (22 early HCC, 15 advanced HCC, and 33
cirrhosis cases) were analyzed, with Hp purified from 20 μL
of serum in each patient, and 140 sets of mass spectrometry (MS) data
were collected using liquid chromatography coupled with electron-transfer
high-energy collisional dissociation tandem MS (LC-EThcD-MS/MS) for
quantitative analysis on a novel software platform, Byos. Differential
quantitation analysis revealed that five N-glycopeptides at sites
N184 and N241 were significantly elevated during the progression from
NASH cirrhosis to HCC (p < 0.05). Receiver operating
characteristic (ROC) curve analysis demonstrated that the N-glycopeptides
at sites N184 and N241 bearing a monofucosylated triantennary glycan
A3G3F1S3 had the best diagnostic performance in detection of early
NASH HCC, area under the curve (AUC) = 0.733 and 0.775, respectively,
whereas α-fetoprotein (AFP) had an AUC of 0.692. When combined
with AFP, the two panels improved the sensitivity for early NASH HCC
from 59% (AFP alone) to 73% while maintaining a specificity of 70%,
based on the optimal cutoff. Two-dimensional (2-D) scatter plots of
the AFP value and N-glycopeptides showed that these N-glycopeptide
markers detected 58% of AFP-negative HCC patients as distinct from
cirrhosis. These site-specific N-glycopeptides could serve as potential
markers for early detection of HCC in patients with NASH-related cirrhosis.