ABSTRACT
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.
ABSTRACT
Due to their unique glycan composition and linkage, protein glycosylation plays significant roles in cellular function and is associated with various diseases. For comprehensive characterization of their extreme structural complexity occurring in >50% of human proteins, time-consuming multi-step enrichment of glycopeptides is required. Here we report zwitterionic n-dodecylphosphocholine-functionalized magnetic nanoparticles (ZIC-cHILIC@MNPs) as a highly efficient affinity nanoprobe for large-scale enrichment of glycopeptides. We demonstrate that ZIC-cHILIC@MNPs possess excellent affinity, with 80-91% specificity for glycopeptide enrichment, especially for sialylated glycopeptide (90%) from biofluid specimens. This strategy provides rapidity (~10 min) and high sensitivity (<1 µL serum) for the whole enrichment process in patient serum, likely due to the rapid separation using magnetic nanoparticles, fast reaction, and high performance of the affinity nanoprobe at nanoscale. Using this strategy, we achieved personalized profiles of patients with hepatitis B virus (HBV, n = 3) and hepatocellular carcinoma (HCC, n = 3) at the depth of >3000 glycopeptides, especially for the large-scale identification of under-explored sialylated glycopeptides. The glycoproteomics atlas also revealed the differential pattern of sialylated glycopeptides between HBV and HCC groups. The ZIC-cHILIC@MNPs could be a generic tool for advancing the glycoproteome analysis, and contribute to the screening of glycoprotein biomarkers.
