Data Independent Acquisition Mass Spectrometry Enhanced Personalized Glycosylation Profiling of Haptoglobin in Hepatocellular Carcinoma

Aberrant glycosylation
has gained significant interest for biomarker
discovery. However, low detectability, complex glycan structures,
and heterogeneity present challenges in glycoprotein assay development.
Using haptoglobin (Hp) as a model, we developed an integrated platform
combining functionalized magnetic nanoparticles and zwitterionic hydrophilic
interaction liquid chromatography (ZIC-HILIC) for highly specific
glycopeptide enrichment, followed by a data-independent acquisition
(DIA) strategy to establish a deep cancer-specific Hp-glycosylation
profile in hepatitis B virus (HBV, n = 5) and hepatocellular
carcinoma (HCC, n = 5) patients. The DIA strategy
established one of the deepest Hp-glycosylation landscapes (1029 glycopeptides,
130 glycans) across serum samples, including 54 glycopeptides exclusively
detected in HCC patients. Additionally, single-shot DIA searches against
a DIA-based spectral library outperformed the DDA approach by 2–3-fold
glycopeptide coverage across patients. Among the four N-glycan sites
on Hp (N-184, N-207, N-211, N-241), the total glycan type distribution
revealed significantly enhanced detection of combined fucosylated-sialylated
glycans, which were the most dominant glycoforms identified in HCC
patients. Quantitation analysis revealed 48 glycopeptides significantly
enriched in HCC (p < 0.05), including a hybrid
monosialylated triantennary glycopeptide on the N-184 site with nearly
none-to-all elevation to differentiate HCC from the HBV group (HCC/HBV
ratio: 2462 ± 766, p < 0.05). In summary,
DIA-MS presents an unbiased and comprehensive alternative for targeted
glycoproteomics to guide discovery and validation of glyco-biomarkers.