In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics

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O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

TidsskriftAnalytical Chemistry
Udgave nummer10
Sider (fra-til)4343-4351
Antal sider9
StatusUdgivet - 2022

Bibliografisk note

Funding Information:
We thank Louise Rosgaard Duus, Karin Uch Hansen, and Sanae Narimatsu, University of Copenhagen, for their expert help with the cell culture. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (GlycoSkin H2020-ERC; 772735), the European Commission (Imgene H2020 and Remodel), the Lundbeck Foundation (R313-2019-869), the Danish National Research Foundation (DNRF107), The Friis Foundation, The Michelsen Foundation, and the A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal.

Publisher Copyright:
© 2022 The Authors. Published by American Chemical Society.

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