GlycoPCT: Pressure Cycling Technology-Based Quantitative Glycoproteomics Reveals Distinctive N‑Glycosylation in Human Liver Biopsy Samples of Nonalcoholic Fatty Liver Disease

Protein N-glycosylation is vital in the human liver and
influences
functions such as lipid metabolism, apoptosis, and inflammation. However,
site-specific N-glycosylation patterns and variations in liver biopsy
samples between healthy individuals and those with nonalcoholic fatty
liver disease (NAFLD) remain incompletely characterized, primarily
due to the limitations of current clinical glycoproteomic methods,
including a large demand for clinical samples, low efficiency of tissue
protein extraction, and a low recovery rate of intact N-glycopeptides
(IGPs). To address this issue, we developed GlycoPCT, a quantitative
glycoproteomic method based on pressure cycling technology. It enables
efficient recovery of IGPs and accurate analysis of trace liver biopsy
samples. Our research revealed a total of 4,459 unique IGPs and 361
glycans from 758 glycoproteins. High-mannose type, complex type, fucosylation
type, and sialylation type N-glycans were significantly upregulated
in the NAFLD group (p < 0.001, t test). Notably, we also identified 182 upregulated IGPs from 67
proteins (p < 0.05, FC > 1.50) and 108 downregulated
IGPs from 44 proteins (p < 0.05, FC < 0.67)
in the NAFLD group. Furthermore, we highlighted an essential acute
phase glycoprotein, alpha-1-acid glycoprotein 1 (A1TA), which is synthesized
in the liver and plays a significant role in NAFLD progression. These
novel glyco-signatures provide crucial clues for the diagnosis and
pathogenesis of NAFLD.