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Abstract
Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling. [Figure not available: see fulltext.]
Original language | English |
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Pages (from-to) | 1143-1155 |
Number of pages | 13 |
Journal | Journal of the American Society for Mass Spectrometry |
Volume | 27 |
Issue number | 7 |
DOIs | |
Publication status | Published - 1 Jul 2016 |
Keywords
- IgM
- Site-specific glycosylation
- Glycopeptide
- Pentamer
- Hexamer
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Dive into the research topics of 'Site-specific N-glycosylation of recombinant pentameric and hexameric human IgM'. Together they form a unique fingerprint.Projects
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Rapid detection of rare-event cells by SUPER Dots: finding a needle in a haystack
Packer, N., Jin, D., Piper, J., Willows, R., Foote, S., Walsh, B., Dubljevic, V., MQRES, M. & CSC-MQRES (International), C.
12/09/13 → 11/09/16
Project: Research