Differentiation between isomeric triantennary N‐linked glycans by negative ion tandem mass spectrometry and confirmation of glycans containing galactose attached to the bisecting (β1‐4‐GlcNAc) residue in N‐glycans from IgG

Abstract

Negative ion tandem mass spectrometry (MS/MS) spectra of three isomeric triantennary N-linked glycans provided clear differentiation between the isomers and confirmed the occurrence of an isomer that was substituted with galactose on a bisecting GlcNAc (1 --> 4-substituted on the core mannose) residue recently reported by Takegawa et al. from N-glycans released from human immunoglobulin G (IgG). We extend this analysis of human serum IgG to reveal an analogue of the fucosylated triantennary glycan reported by Takegawa et al. together with a third compound that lacked both the sialic acid and the fucose residues. In addition, we demonstrate the biosynthesis of bisected hybrid-type glycans with the galactose modification, with and without core fucose, on the stem cell marker glycoprotein, 19A, expressed in a partially ricin-resistant human embryonic kidney cell line. It would appear, therefore, that this modification of N-linked glycans containing a galactosylated bisecting GlcNAc residue may be more common than originally thought. Negative ion MS/MS analysis of glycans is likely to prove an invaluable tool in the analysis and monitoring of therapeutic glycoproteins.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…The presence of a biantennary, bisected N-glycan containing a galactose residue attached to the bisecting GlcNAc (49), first reported by Takegawa et al (2005) from IgG, has also been identified by Harvey et al (2008b) as part of a study into negative ion MS/MS for the differentiation of isomeric triantennary Effect of culture conditions. Glycan structure inferred by "GlycoMod" (Nam, et al, 2008) Chorionic gonadotropin…”

Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status

“…MS/MS fragmentation described by Harvey (48). In negative mode fragmentation spectra of N-glycans, the D ion arises from the loss of the chitobiose core and the substituents forming the 3-antennae; thus the D ion mass corresponds to the composition of the 6-arm antenna as well as the two remaining branching core mannose residues.…”

Fragmentation of negative ions from N‐linked carbohydrates, Part 4. Fragmentation of complex glycans lacking substitution on the 6‐antenna

“…The spectra (not shown) of complex N-glycans containing two antennae attached to the 6-linked mannose, such as IV and V, contain an additional ion at [D-36] − of similar relative abundance. [15] This ion, which is sometimes also present in the spectra of glycans containing only one branch at the 6-position is usually of only low abundance. Monoantennary Nlinked glycans with the same composition as those shown in Fig.…”

“…The 2,4-di-substituted 3-antenna in compound VIII gives rise to a prominent ion at m/z 831 (termed ion E) as is seen in the spectra of the 3-branched triantennary glycans (XI; Fig. 7c) [15] but which is absent in the spectrum of the normal biantennary isomer (III; Fig. 6b).…”