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

Protein glycosylation presents profound analytical challenges due to its structural heterogeneity, yet robust workflows capable of resolving glycosite- and glycan-level information within complex clinical cohorts remain limited. To address this gap, we developed an integrated dual-layer N-glycoproteomic strategy that combines high-coverage site-specific N-glycosylation profiling with intact N-glycopeptide analysis to preserve glycan features. Type 2 diabetes mellitus complicated with coronary heart disease (T2DM&CHD) represents a severe yet understudied comorbidity, and the scarcity of well-characterized patient cohorts underscores the analytical value of our uniquely curated dataset. Applying this strategy to serum samples from healthy controls (n = 43), T2DM (n = 39), CHD (n = 37), and T2DM&CHD (n = 46) generated 497 N-glycosites from 781 site-specific N-glycopeptides and 133 N-glycosites from 341 intact N-glycopeptides, demonstrating the complementary breadth and depth attained by integrating the two layers. The integrated analysis revealed extensive glycoproteome remodeling in T2DM&CHD, characterized by increased microheterogeneity driven by altered sialylation and fucosylation. Site-specific changes in apolipoprotein B, particularly at N185, N1523, N2239, and N3411, showed stage-dependent patterns associated with lipid dysregulation and vascular injury. Functional enrichment indicated that T2DM amplifies inflammation-related glycosylation disturbances that predispose to cardiovascular complications. Correlation with glycosyltransferase expression further underscored the regulatory relevance of glycan biosynthesis. Together, our results develop an extendable analytical framework for comprehensive serum glycoproteome characterization and provide a mechanistic resource for biomarker discovery in metabolic-cardiovascular comorbidity.