Site-Specific N‑Glycosylation Analysis of Human Immunoglobulin E

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.