Dataset Information

Enabling Epitope Analysis of Glycoproteins via HDX-MS Using Antigens with Uni-formed N-linked Glycans

ABSTRACT: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is widely used for epitope analysis of the antibod-ies. However, epitope analysis of glycoproteins is challenging because of the heterogeneity of attached N-linked glycans. Recent studies have reported methods where N-linked glycans were removed at low pH follow-ing hydrogen deuterium exchange reactions, but the methods for glycoproteins remain controversial. Here, we demonstrate the utility of using antigens with uniformed N-linked glycans in glycoprotein epitope analysis by HDX-MS. By treating HEK293 cells with kifunensine, we were able to prepare antigens mainly with N-linked high-mannose-type glycans. Analysis of epitopes of a monoclonal antibody S309 using antigens prepared with this method allowed us to identify an epitope that included the previously reported N-linked glycan attachment sites for this antibody. We propose that using antigens with uniformed glycans should be effective for epitope analysis of glycoproteins, such as virus spike proteins covered by glycan shields. Moreover, we believe that this approach accelerates research on vaccines and neutralizing antibodies against viruses that escape host immunity through glycan shields or mutations.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Takeshi Ota

PROVIDER: PXD056361 | JPOST Repository | Sat Sep 27 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	20240411_RBD_Wuhan_AbACE2_UptakeSummaryTable.csv	Csv
	20240415_RBD_Wuhan_peptidemapping.csv	Csv
	20240627_RBD_Wuhan-AbS309_n3_UptakeSummaryTable.csv	Csv
	Fig1_peptidemapping.zip	Other
	Fig2_ACE2.zip	Other

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Project description:Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS 2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Dataset Information

Enabling Epitope Analysis of Glycoproteins via HDX-MS Using Antigens with Uni-formed N-linked Glycans

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