Integrated glycoproteomics reveals site-specific N-glycosylation defects in phosphomannomutase two congenital disorder of glycosylation.

The routine diagnostic pathway for congenital disorders of glycosylation (CDG) relies on the analysis of carbohydrate-deficient transferrin in serum, a test that incompletely captures defects in N-linked glycosylation and may also yield false-negative results even in classical N-glycosylation disorders such as PMM2-CDG. To explore molecular features of PMM2-CDG pathogenesis and identify potential biomarker candidates, we applied an integrated semiquantitative LC-MS/MS glycoproteomic workflow to a well-characterized cohort of individuals with PMM2-CDG (n = 7), alongside controls (n = 9). The workflow combines the analysis of N-glycopeptides and corresponding non-glycosylated peptides in serum and plasma. Distinct disease-associated glycosylation patterns were observed in PMM2-CDG patients. Elevated levels of immature (paucimannose-like) N-glycans were detected at specific sites, including GlcNAc(2)Man(4) at Asn-85 of complement component 3, GlcNAc(2)Man(3) at Asn-869 of α-2-macroglobulin, and GlcNAc(2)Man(5) at Asn-226 of complement component 4. In parallel, approximately 50 peptides with unoccupied N-glycosylation sequons, including peptides from transferrin, were recurrently identified in CDG patient samples, indicating widespread reductions in N-glycosylation site occupancy. Together, this small cohort-based glycoproteomic analysis reveals coherent, site-specific disruptions in protein glycosylation that reflect underlying defects in mannose metabolism in PMM2-CDG. These molecular features define candidate glycophenotypes with potential utility as biomarkers for CDG subtype discrimination, disease stratification, and assessment of disease severity in future studies.