Integrated N- and O-Glycoproteomics Reveals Site-Specific Glycosylation Dysregulation in Alzheimer’s Disease

Abstract Alzheimer’s disease (AD) remains a global health challenge, in part due to the lack of reliable blood-based biomarkers for early detection and disease monitoring. Protein glycosylation critically regulates the structure and function of circulating macromolecules, yet integrated site-specific evaluation of both N- and O-glycosylation across the AD continuum remains poorly defined. Here, we performed integrated intact N- and O-glycoproteomic profiling of human serum from normal controls, patients with mild cognitive impairment (MCI), and patients with AD and identified 2,433 intact N-glycopeptides and 3,864 intact O-glycopeptides. Quantitative analysis revealed that N-glycosylation changes emerged earlier, highlighted by a marked reduction in site-specific Fc-region glycans on immunoglobulin heavy constant gamma 1 (IGHG1, Asn180) at the MCI stage, suggesting early perturbation of immune regulation. In contrast, O-glycosylation changes were broader and more progressive, exemplified by increased C-terminal O-glycoforms of apolipoprotein E (APOE) from controls to patients with MCI and AD, consistent with dysregulated lipid transport and receptor-associated functions. Together, these findings establish an integrated serum glycoproteomic atlas across the AD continuum and nominate IGHG1 and APOE glycoforms as candidate group-associated glycopeptide features. This study highlights intact glycopeptide profiling as a powerful strategy for site-specific biomarker discovery and hypothesis-generating mechanistic insight in AD progression.