Site‐specific analysis of the N‐glycans on murine polymeric immunoglobulin A using liquid chromatography/electrospray mass spectrometry

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Abstract
The site‐specific distribution of oligosaccharides on murine polymeric immunoglobulin A (pIgA) consisting of two or more immunoglobulin A (IgA) antibodies connected through J‐chain was analysed by liquid chromatography/electrospray mass spectrometry. Glycopeptides from pIgA were localized in a reversed‐phase tryptic peptide chromatogram by collision excitation scanning and their amino acid sequences determined by electrospray tandem mass spectrometry and Edman degradation. Two glycosylation sites on IgA and a single glycosylation site on J‐chain were identified. Using biosynthetic constraints on carbohydrate structures, molecular mass information on the glycan moieties of the glycopeptides was translated into specific carbohydrate structure proposals. The glycopeptide incorporating the single glycosylation site at Asn49 in J‐chain carried fucosylated and non‐fucosylated di‐and triantennary N‐acetyllactosamine type carbohydrate chains terminated by N‐acetyl‐ and/or N‐glycolylneuraminic acid residues. The glycosylation site in the IgA heavy chain at Asn446 contained two oligomannose‐type carbohydrate chains, one carrying five and the other six mannose residues. To the other glycosylation site in the IgA heavy chain at Asn155 one oligomannose structure, hybrid structures with the lactosamine branch terminated by either an additional galactose residue, N‐glycolylneuraminic acid or N‐acetylneuraminic acid, and non‐fucosylated N‐acetyllactosamine‐type structures carrying the same terminating residues were attached. This glycosylation site was present in two separate glycopeptides differing only in their degree of carboxymethylation and yielding identical oligosaccharide distributions, thus providing additional confidence in the assignment method.