Comprehensive analysis of O-glycosylation of amyloid precursor protein (APP) using targeted and multi-fragmentation MS strategy

Background: The aberrant proteolytic processing of amyloid precursor protein (APP) into amyloid beta peptide (A beta) in brain is a critical step in the pathogenesis of Alzheimer's disease (AD). As an O-glycosylated protein, O-glycosylation of APP is considered to be related to A beta generation. Therefore, comprehensive analysis of APP O-glycosylation is important for understanding its functions. Methods: We developed a Targeted MS approach with Multi-Fragmentation techniques (TMMF strategy), and successfully characterized O-glycosylation profiling of APP695 expressed in HEK-293 T cells. We calculated relative abundance of glycopeptides with various O-glycosites and O-glycans, and further investigated the alteration of APP O-glycosylation upon TNF-alpha treatment. Results: A total of 14 O-glycosites were identified on three glycopeptides of APP, and at least four O-glycans including GalNAc (Tn antigen), core 1, and mono-/di-sialylated core 1 glycans were determinant at the residues of Thr576 and Thr577. We found a dense cluster of truncated O-glycans on the region nearby beginning of E2 domain and high abundance of sialylated O-glycans on the region close to beta-cleavage site. Moreover, we also observed that TNF-alpha could upregulate the expression of APP and the truncated O-glycans on APP in HEK-293 T cell. Conclusion: Our study established an intact O-glycopeptide MS analysis strategy for APP O-glycopeptide identification with enhanced fragmentation efficiency and detection sensitivity. These results provide a comprehensive O-glycosylation map of APP expressed in HEK-293 T cell. General significance: The accurate O-glycosites and O-glycan structures on APP may lead to a better understanding of the roles O-glycosylation plays in the processing and functions of APP.

Automated summary

This study established a comprehensive O-glycopeptide mass spectrometry analysis strategy for APP O-glycopeptide identification, successfully identifying 14 O-glycosites on APP695 expressed in HEK-293 T cells. The research found at least four O-glycans present at specific residues on APP, providing valuable insights into the O-glycosylation patterns of APP.