MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation
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...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-08-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fimmu.2020.02049/full |
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| author | Thomas Sénard Andrea F. G. Gargano Andrea F. G. Gargano David Falck Steven W. de Taeye Steven W. de Taeye Theo Rispens Gestur Vidarsson Manfred Wuhrer Govert W. Somsen Elena Domínguez-Vega |
| author_facet | Thomas Sénard Andrea F. G. Gargano Andrea F. G. Gargano David Falck Steven W. de Taeye Steven W. de Taeye Theo Rispens Gestur Vidarsson Manfred Wuhrer Govert W. Somsen Elena Domínguez-Vega |
| author_sort | Thomas Sénard |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T20:21:08Z |
| format | Article |
| id | doaj.art-0351159d2b654d0eae826055873c3847 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-12T20:21:08Z |
| publishDate | 2020-08-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Immunology |
| spelling | doaj.art-0351159d2b654d0eae826055873c38472022-12-22T00:13:15ZengFrontiers Media S.A.Frontiers in Immunology1664-32242020-08-011110.3389/fimmu.2020.02049565585MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-GlycosylationThomas Sénard0Andrea F. G. Gargano1Andrea F. G. Gargano2David Falck3Steven W. de Taeye4Steven W. de Taeye5Theo Rispens6Gestur Vidarsson7Manfred Wuhrer8Govert W. Somsen9Elena Domínguez-Vega10Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, NetherlandsVan ’t Hoff Institute for Molecular Sciences, University of Amsterdam Analytical Chemistry Group, Amsterdam, NetherlandsAmsterdam Institute for Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, NetherlandsCenter for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, NetherlandsDepartment of Experimental Immunohematology, Sanquin Research, Amsterdam, NetherlandsDepartment of Immunopathology, Sanquin Research, Amsterdam, NetherlandsDepartment of Immunopathology, Sanquin Research, Amsterdam, NetherlandsDepartment of Experimental Immunohematology, Sanquin Research, Amsterdam, NetherlandsCenter for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, NetherlandsAmsterdam Institute for Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, NetherlandsCenter for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, NetherlandsCurrent 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.https://www.frontiersin.org/article/10.3389/fimmu.2020.02049/fullimmunoglobulin Gallotypesfragment crystallizableN-glycosylationpost-translational modificationsmass spectrometry |
| spellingShingle | Thomas Sénard Andrea F. G. Gargano Andrea F. G. Gargano David Falck Steven W. de Taeye Steven W. de Taeye Theo Rispens Gestur Vidarsson Manfred Wuhrer Govert W. Somsen Elena Domínguez-Vega MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation Frontiers in Immunology immunoglobulin G allotypes fragment crystallizable N-glycosylation post-translational modifications mass spectrometry |
| title | MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation |
| title_full | MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation |
| title_fullStr | MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation |
| title_full_unstemmed | MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation |
| title_short | MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation |
| title_sort | ms based allotype specific analysis of polyclonal igg fc n glycosylation |
| topic | immunoglobulin G allotypes fragment crystallizable N-glycosylation post-translational modifications mass spectrometry |
| url | https://www.frontiersin.org/article/10.3389/fimmu.2020.02049/full |
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