title: Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.
abstract: The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.
authors.affiliation: From the ‡Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands; §Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
authors.forename: Albert
authors.initials: A
authors.lastname: Bondt
authors.affiliation: §Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; ¶Department of Rheumatology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
authors.forename: Yoann
authors.initials: Y
authors.lastname: Rombouts
authors.affiliation: §Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
authors.forename: Maurice H J
authors.initials: MH
authors.lastname: Selman
authors.affiliation: §Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
authors.affiliation: §Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;
authors.forename: Karli R
authors.initials: KR
authors.lastname: Reiding
authors.affiliation: From the ‡Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands;
authors.forename: Johanna M W
authors.initials: JM
authors.lastname: Hazes
authors.affiliation: From the ‡Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands;
authors.forename: Radboud J E M
authors.initials: RJ
authors.lastname: Dolhain
authors.affiliation: §Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands; ‖Division of BioAnalytical Chemistry, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands; **Department of Molecular Cell Biology and Immunology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands m.wuhrer@lumc.nl.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D005260:Female; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000097:blood*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification; D049590:Postpartum Period; D011247:Pregnancy; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D006023:Glycoproteins
chemical_list: D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D019158:N-Acetylneuraminic Acid; D011134:Polysaccharides; C046179:glycosylated IgG; D006023:Glycoproteins