Integrative Analysis of Fucosylated Tetra Glycoforms in Hepatocellular Carcinoma: A NanoLC-PRM-MS/MS and Machine Learning Approach

Hepatocellular carcinoma
(HCC), commonly associated with cirrhosis,
is a major cause of cancer-related mortality due to its poor prognosis.
Herein, we investigated fucosylated glycoforms of serum haptoglobin
(Hp) as potential biomarkers for HCC of metabolic associated dysfunction
associated liver disease (MASLD) and alcohol related liver disease
(ALD) etiologies. We analyzed 119 patient samples, including 60 with
cirrhosis and 59 with HCC. Isolated Hp protein was digested using
trypsin and Glu-C, and site-specific N-glycans were
quantified using PRM with LC-HCD-MS/MS. Differential analysis revealed
significant variations in fucosylated tetra-antennary glycoforms at
N241(VVLHPN241YSQVD and VVLHPN241YSQVDIGLIK),
particularly in distinguishing cirrhosis and HCC (P < 0.05). A combined analysis of identified tetra-antennary fucosylated
markers, along with AFP, gender, and age, demonstrated improved AUC.
Tetra-antennary glycoforms exhibited an AUC of 0.871 (95% CI: 0.80–0.93)
when incorporated into the AFP + age + gender + marker panel compared
to AFP alone (0.756) with a sensitivity of 0.763 at a specificity
of 0.80. 3-fold cross validation was further used to assess the performance
of the optimal biomarker panel. Thus, a combination of fucosylated
tetra-antennary glycoforms may serve as important markers for distinguishing
HCC from cirrhosis.