Differentiation between isomeric triantennary N-linked glycans by negative ion tandem mass spectrometry and confirmation of glycans containing galactose attached to the bisecting (beta1-4-GlcNAc) residue in N-glycans from IgG.
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.
Harvey
David J
DJ
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK. david.harvey@bioch.ox.ac.uk
Crispin
Max
M
Scanlan
Chris
C
Singer
Bernhard B
BB
Lucka
Lothar
L
Chang
Veronica T
VT
Radcliffe
Catherine M
CM
Thobhani
Smita
S
Yuen
Chun-Ting
CT
Rudd
Pauline M
PM
D000838:Anions; D014408:Biomarkers, Tumor / Q000737:chemistry*; D005690:Galactose / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D007536:Isomerism; D017350:N-Acetylgalactosaminyltransferases; D009842:Oligopeptides / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000838:Anions; D014408:Biomarkers, Tumor; D007074:Immunoglobulin G; D009842:Oligopeptides; D011134:Polysaccharides; D017350:N-Acetylgalactosaminyltransferases; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; D005690:Galactose