Dual-layer N-glycoproteomics enhances resolution of serum glycosylation signatures in type 2 diabetes mellitus complicated with coronary heart disease.

The dual-layer N-glycoproteomic strategy generated high-coverage glycosylation data from serum samples across four clinical groups.

• Serum samples were collected from healthy controls (n=43), T2DM (n=39), CHD (n=37), and T2DM&CHD (n=46).
• The site-specific N-glycosylation profiling layer generated 497 N-glycosites from 781 site-specific N-glycopeptides.
• The intact N-glycopeptide analysis layer generated 133 N-glycosites from 341 intact N-glycopeptides.
• The two analytical layers demonstrated complementary breadth and depth when integrated.

T2DM complicated with CHD exhibited extensive glycoproteome remodeling characterized by increased microheterogeneity.

• The remodeling in T2DM&CHD was driven by altered sialylation and fucosylation patterns.
• The integrated dual-layer analysis was required to fully resolve these glycan-level changes.
• Microheterogeneity refers to multiple distinct glycan structures occupying the same glycosylation site.

Site-specific changes in apolipoprotein B showed stage-dependent patterns associated with lipid dysregulation and vascular injury.

• Specific glycosylation changes were identified at apolipoprotein B sites N185, N1523, N2239, and N3411.
• These changes exhibited stage-dependent patterns across the disease groups studied (healthy controls, T2DM, CHD, T2DM&CHD).
• The site-specific changes were associated with lipid dysregulation and vascular injury.

Functional enrichment analysis indicated that T2DM amplifies inflammation-related glycosylation disturbances that predispose to cardiovascular complications.

• Enrichment analysis was applied to differentially glycosylated proteins identified across disease groups.
• T2DM-associated glycosylation disturbances were linked to inflammatory pathways.
• These disturbances were interpreted as predisposing factors for subsequent cardiovascular complications.

Correlation between glycoproteome changes and glycosyltransferase expression underscored the regulatory relevance of glycan biosynthesis.

• Glycosyltransferase expression data were correlated with observed glycosylation alterations.
• The correlation supported a biosynthetic regulatory basis for the observed glycoproteome remodeling.
• This analysis linked upstream enzymatic regulation to the downstream glycan structural changes detected in patient serum.

The dual-layer strategy demonstrated that integrating site-specific and intact N-glycopeptide analyses provides complementary information not achievable by either approach alone.

• Site-specific profiling provided high coverage of glycosylation sites (497 N-glycosites).
• Intact N-glycopeptide analysis preserved glycan features to resolve glycan composition (341 intact N-glycopeptides from 133 N-glycosites).
• The strategy was described as an 'extendable analytical framework for comprehensive serum glycoproteome characterization.'