TY - JOUR

T1 - In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics

AU - de Haan, Noortje

AU - Narimatsu, Yoshiki

AU - Aasted, Mikkel Koed Møller

AU - Larsen, Ida S. B.

AU - Marinova, Irina N.

AU - Dabelsteen, Sally

AU - Vakhrushev, Sergey Y.

AU - Wandall, Hans H.

N1 - Publisher Copyright: © 2022 The Authors. Published by American Chemical Society.

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

U2 - 10.1021/acs.analchem.1c05068

DO - 10.1021/acs.analchem.1c05068

M3 - Journal article

C2 - 35245040

AN - SCOPUS:85126311450

VL - 94

SP - 4343

EP - 4351

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 10

ER -