The N‑Glycoproteomic Landscape of the Lung in Monocrotaline-Induced Pulmonary Arterial Hypertension.

Protein N-glycosylation plays critical roles in controlling cell function, yet its specific contributions to pulmonary arterial hypertension (PAH) remain poorly understood. Here, we performed an integrated proteomic and site-specific N-glycoproteomic analysis of lung tissues from a monocrotaline-induced PAH rat model. Using quantitative LC-MS/MS with glycoprotein enrichment, we identified 7,048 proteins in the proteome, including 1,302 differentially expressed proteins and 1,918 N-glycosylation sites across 764 glycoproteins in the N-glycoproteome. 320 glycosylation sites (from 260 glycoproteins) showed significant dysregulation. Glycoproteomic motif analysis confirmed the canonical N-X-S/T sequon, with enriched localization in plasma membrane and extracellular proteins. Bioinformatics revealed ribosome-related pathways were significantly downregulated, whereas lysosomal pathways were upregulated. Key N-glycoproteomic alterations involved integrin binding, ECM-receptor interaction, and ferroptosis. Integrated multiomics analysis of 7,136 proteome and 2,897 N-glycoproteome proteins identified 805 overlapping candidates, of which 387 exhibited concordant upregulation in lysosome, TGF-β signaling, and mineral absorption pathways. We confirmed that VCAM1 and AMBP were increased in the lungs of PAH rats by Western blot and immunofluorescence. Our study provides the first system-wide view of the N-glycoproteomic landscape of the lung in monocrotaline-induced PAH rats, revealing the involvement of N-glycosylation in ECM dysfunction, adhesion pathways, and lysosomal dysregulation, and suggests novel glycoprotein-centered targets for therapeutic intervention.