title: Site-specific N-glycosylation analysis of human immunoglobulin e.
abstract: Immunoglobulin E (IgE) is a heterodimeric glycoprotein involved in antiparasitic and allergic immune reactions. IgE glycosylation is known to exhibit significant interindividual variation, and several reports have indicated its relevance in determining IgE activity. Here, we present site-specific glycosylation analysis of IgE from three different sources: IgE from the serum of a hyperimmune donor, from the pooled serum of multiple nondiseased donors, and from the pooled serum of 2 patients with IgE myeloma. The heavy chains were isolated and digested with either trypsin, proteinase K, or chymotrypsin, which permitted coverage of all seven potential N-glycosylation sites. The resulting (glyco-)peptides were analyzed by nano-reversed-phase-LC-MS/MS and MALDI-TOF/TOF-MS/MS. Site Asn264 was shown to be unoccupied. In all three samples, site Asn275 contained exclusively oligomannosidic structures with between 2 and 9 mannoses, whereas sites Asn21, Asn49, Asn99, Asn146, and Asn252 contained exclusively complex-type glycans. For the nonmyeloma IgE, the majority of these glycans were biantennary and core-fucosylated and contained one or two terminal N-acetylneuraminic acids. In contrast, myeloma IgE showed a higher abundance of triantennary and tetraantennary glycan structures and a low abundance of species with a bisecting N-acetylglucosamine. Our approach allows comparison of the glycosylation of IgE samples in a site-specific manner.
authors.affiliation: Center for Proteomics and Metabolomics, ‡Department of Rheumatology, Leiden University Medical Center , 2300 RC Leiden, The Netherlands.
authors.forename: Rosina
authors.initials: R
authors.lastname: Plomp
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hensbergen
authors.forename: Yoann
authors.initials: Y
authors.lastname: Rombouts
authors.forename: Gerhild
authors.initials: G
authors.lastname: Zauner
authors.forename: Irina
authors.initials: I
authors.lastname: Dragan
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D007073:Immunoglobulin E