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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 10; p. 129954
• Main Authors: Shi, Jingjing, Ku, Xin, Zou, Xia, Hou, Jingli, Yan, Wei, Zhang, Yan
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.10.2021
• Subjects: Alzheimer disease; Alzheimer's disease; amyloid; antigens; APP; brain; detection limit; glycopeptides; glycosylation; Intact O-glycopeptides; Multiple fragmentation MS analysis; O-glycosylation; pathogenesis; polysaccharides; proteolysis; VVA; APP; AD; Neu5Ac; Multiple fragmentation MS analysis; HCD; O-glycosylation; ppGalNAcTs; GalNAc; Aβ; LC-MS/MS; Gal; ETD; TNF-α; TMMF; Intact O-glycopeptides; HexNAc; Hex; GlcNAc; qPCR; Alzheimer's disease; CID
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2021.129954

The aberrant proteolytic processing of amyloid precursor protein (APP) into amyloid β peptide (Aβ) 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 Aβ generation. Therefore, comprehensive analysis of APP O-glycosylation is important for understanding its functions. 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-α treatment. 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 β-cleavage site. Moreover, we also observed that TNF-α could upregulate the expression of APP and the truncated O-glycans on APP in HEK-293 T cell. 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. 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. •A novel intact O-glycopeptide MS analysis tool (TMMF strategy) with high detection efficiency.•TMMF strategy provides a comprehensive O-glycosylation map of APP in HEK-293 T cell.•The O-glycosylation level of APP could be upregulated by TNF-α stimulation.