Selective identification of α-galactosyl epitopes in N-glycoproteins using characteristic fragment ions from higher-energy collisional dissociation

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Title
Selective identification of α-galactosyl epitopes in N-glycoproteins using characteristic fragment ions from higher-energy collisional dissociation
Author(s)
H K Lee; Dae In Ha; Jeong Gu Kang; G W Park; J Y Lee; K Cho; Su Bin Moon; J H Shin; Yong-Sam Kim; H J An; J Y Kim; J S Yoo; Jeong Heon Ko
Bibliographic Citation
Analytical Chemistry, vol. 92, no. 19, pp. 13144-13154
Publication Year
2020
Abstract
The α-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 α-galactosyl structure in biopharmaceuticals or organ transplants. Due to an identical glycan composition and molecular mass between α-galactosyl N-glycans and hybrid/high-mannose-type N-glycans, it is challenging to characterize α-galactosyl epitopes in N-glycoproteins using mass spectrometry. Here, we describe a method to identify α-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 [YHM] 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 (Hex2HexNAc1) ion to m/z 366.14 (Hex1HexNAc1) ion (Im/z528/Im/z366). The measure of [Im/z528/Im/z366 > 0.3] enabled a clear-cut determination of α-galactosyl N-glycopeptides with high accuracy. In Ggta1 knockout mice, we could not find any α-galactosyl N-glycoproteins identified in WT mice plasma. Using this method, we could screen for α-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 α-galactosyl epitopes in pharmaceuticals and complex biological samples.
ISSN
0003-2700
Publisher
Amer Chem Soc
DOI
http://dx.doi.org/10.1021/acs.analchem.0c02276
Type
Article
Appears in Collections:
Division of Biomedical Research > Genome Editing Research Center > 1. Journal Articles
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