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MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation

Authors :
Thomas Sénard
Andrea F. G. Gargano
David Falck
Steven W. de Taeye
Theo Rispens
Gestur Vidarsson
Manfred Wuhrer
Govert W. Somsen
Elena Domínguez-Vega
Landsteiner Laboratory
AII - Inflammatory diseases
Supramolecular Separations (HIMS, FNWI)
BioAnalytical Chemistry
AIMMS
Source :
Frontiers in immunology, 11:2049. Frontiers Media S.A., Frontiers in Immunology, 11:2049. Frontiers Media S.A., Frontiers in Immunology, Vol 11 (2020), Sénard, T, Gargano, A F G, Falck, D, de Taeye, S W, Rispens, T, Vidarsson, G, Wuhrer, M, Somsen, G W & Domínguez-Vega, E 2020, ' MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation ', Frontiers in Immunology, vol. 11, 2049, pp. 1-13 . https://doi.org/10.3389/fimmu.2020.02049, Frontiers in Immunology, 11. FRONTIERS MEDIA SA, Frontiers in Immunology, Frontiers in Immunology, 11:2049, 1-13. Frontiers Media S.A.
Publication Year :
2020
Publisher :
Frontiers Media SA, 2020.

Abstract

Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the N-glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead CH2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants.

Details

ISSN :
16643224
Volume :
11
Database :
OpenAIRE
Journal :
Frontiers in Immunology
Accession number :
edsair.doi.dedup.....37cf15b625d5dd9b5827662e641a853e