Time grid-based isomer specific N-glycan analysis and detection of bisecting Lewis X in human brain

Helm, J.; Gruenwald-Gruber, C.; Thader, A.; Urteil, J.; Fuehrer, J.; Stenitzer, D.; Maresch, D.; Neumann, L.; Pabst, M.; Altmann, F.

doi:10.1101/2021.04.14.439640

bioRxiv · 10.1101/2021.04.14.439640

Time grid-based isomer specific N-glycan analysis and detection of bisecting Lewis X in human brain

Helm, J.·Gruenwald-Gruber, C.·Thader, A.·Urteil, J.·Fuehrer, J.·Stenitzer, D.·Maresch, D.·Neumann, L.·Pabst, M.·Altmann, F.

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Abstract

The importance of protein glycosylation in the biomedical field demands for methods capable of resolving and identifying isomeric structures of N-glycans. However, the unambiguous identification of isomeric structures from complex mixtures is currently not reasonably realized even by the most sophisticated approaches. Here we present a novel approach which uses stable isotope labelled reference N-glycans to establish a retention time grid (glyco-TiGr) on porous graphitized carbon. This furthermore enables retention as the primary criterion for the structural assignment of isomeric N-glycans. Moreover, we biosynthesized forty natural isomers of the fundamental N-glycan type consisting of five hexoses, four N-acetylhexosamines and one fucose residue. Nearly all of these isomers occupied unique positions on the retention time grid. Reference glycan assisted retention time determination with deci-minute accuracy narrowed the assignment space to very few, often only one possible glycan isomer. Application of the glyco-TiGr approach revealed yet undescribed isomers of Lewis x determinants in multimeric human IgA and hybrid type N-glycans in human brain with galactose and even fucose linked to the bisecting N-acetylglucosamine. Thus, the brain N-glycome displayed a degree of sophistication commensurate with this organs role.

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Helm, J., Gruenwald-Gruber, C., Thader, A., Urteil, J., Fuehrer, J., Stenitzer, D., Maresch, D., Neumann, L., Pabst, M., Altmann, F.. 2021-04-14. Time grid-based isomer specific N-glycan analysis and detection of bisecting Lewis X in human brain. https://doi.org/10.1101/2021.04.14.439640

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