Selective Identification Of A-Galactosyl Epitopes In N-Glycoproteins Using Characteristic Fragment Ions From Higher-Energy Collisional Dissociation

The alpha-galactosyl epitope is a terminal N-glycan moiety of glycoproteins found in mammals except in humans, and thus, it is recognized as an antigen that provokes an immunogenic response in humans. Accordingly, it is necessary to analyze the alpha-galactosyl structure in biopharmaceuticals or organ transplants. Due to an identical glycan composition and molecular mass between alpha-galactosyl N-glycans and hybrid/high-mannose-type N-glycans, it is challenging to characterize alpha-galactosyl epitopes in N-glycoproteins using mass spectrometry. Here, we describe a method to identify alpha-galactosyl N-glycopeptides in mice glycoproteins using liquid chromatography with tandem mass spectrometry with higher-energy collisional dissociation (HCD). The first measure was an absence of [Y-HM] ion peaks in the HCD spectra, which was exclusively observed in hybrid and/or high-mannose-type N-glycopeptides. The second complementary criterion was the ratio of an m/z 528.19 (Hex(2)HexNAc(1)) ion to m/z 366.14 (Hex(1)HexNAc(1)) ion (I-m/z528/I-m/z366). The measure of [I-m/z528/I-m/z366 > 0.3] enabled a clear-cut determination of alpha-galactosyl N-glycopeptides with high accuracy. In Ggta1 knockout mice, we could not find any alpha-galactosyl N-glycoproteins identified in WT mice plasma. Using this method, we could screen for alpha-galactosyl N-glycoproteins from mice spleen, lungs, and plasma samples in a highly sensitive and specific manner. Conclusively, we suggest that this method will provide a robust analytical tool for determination of alpha-galactosyl epitopes in pharmaceuticals and complex biological samples.