A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase

Kameyama, Akihiko; Tin, Wai Wai Thet; Toyoda, Masashi; Sakaguchi, Midori

doi:10.1016/j.bbrc.2019.03.144

Cited by 32 publications

(34 citation statements)

References 24 publications

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“… 9 Importantly, the nonreductive β-elimination used for the liberation of the O -linked glycans allows for functionalization of the reducing ends of the glycans. 9 We exploited this feature by labeling the released O -glycans with 2-AB, which facilitated isomer separation on a C18 nanoflow LC column.…”

Section: Resultsmentioning

confidence: 99%

“…During the optimization of the reaction, samples were incubated with final concentrations of 20% hydroxylamine and 20% DBU, 10% hydroxylamine and 40% DBU, or 0% hydroxylamine and 20% DBU for 1 h at 37 °C. 9 The pH of these conditions was determined using pH paper to increase between 11 and 14 with decreasing concentrations of hydroxylamine, independent of the DBU concentration. The O -glycans were recovered from the membrane by 2 min centrifugation at 1000 g , and 1 mL of acetonitrile (ACN) containing 2 mg of magnetic hydrazide beads (MagSi-S Hydrazide beads 1 μm, magtivio B.V., Nuth, The Netherlands) was added.…”

Section: Experimental Sectionmentioning

confidence: 99%

“…Recently, great progress was made in the chemical release and labeling of O -glycans, keeping glycan degradation to a minimum. 9 , 10 While these developments are important, it remains challenging to separate and characterize O -glycan isomers and to integrate the analysis in a high-throughput setup of complex samples often with limited tissue material available for the analysis.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Experimental Sectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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“…Traditional analysis for defining the O‐glycan glycome from cultured cells involves the chemical release of glycans from glycoproteins or glycopeptides by strong base treatment (Current Protocols article: Fukuda, 1995; Kameyama et al., 2019; Morelle & Michalski, 2007; Wilkinson & Saldova, 2020). Although this approach is useful when relatively large amounts of cells are available, the inefficient release of glycans coupled with glycan degradation due to exposure of reducing glycans to strong base results in low sensitivity and production of glycan fragments of O‐glycans that increases the complexity of interpreting results.…”

Section: Commentarymentioning

confidence: 99%

“…In addition, functional O‐glycomics is more challenging due to lack of endo‐glycosidases that can universally release O‐glycans from glycoproteins, in contrast to N‐glycomics, which is facilitated by available enzymes PNGase F and PNGase A that can release all types of N‐glycans from glycoproteins. In the analysis of O‐glycans, their release from glycoproteins often relies on chemical methods such as β‐elimination (Current Protocols article: Fukuda, 1995; Kameyama, Thet Tin, Toyoda, & Sakaguchi, 2019; Wilkinson & Saldova, 2020), which have many limitations, such as non‐specificity and low efficiency, and often the requirement of large amounts of material to identify minor structures. Quantitative analysis of O‐glycans or quantitative O‐glycomics is an unmet demand (Mechref, Hu, Desantos‐Garcia, Hussein, & Tang, 2013).…”

Section: Introductionmentioning

confidence: 99%

Cellular O‐Glycome Reporter/Amplification (CORA): Analytical and Preparative Tools to Study Mucin‐Type O‐Glycans of Living Cells

Kudelka

Chernova

et al. 2021

Current Protocols

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Mucin-type O-glycosylation (O-glycans, O-glycome) is among the most biologically important post-translational modification in glycoproteins but O-glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O-glycome reporter/amplification (CORA), which uses Oglycan precursors, benzyl-α-GalNAc (Bn-α-GalNAc) or azido-Bn-α-GalNAc (N 3 -Bn-α-GalNAc), as surrogates of protein O-glycosylation. Living cells metabolically convert these precursors to all types of O-GalNAc glycans representative of the cells' capabilities. The amplification and secretion of the O-glycome products greatly facilitates their analysis and functional studies. Here we describe protocols for analytical and preparative applications.

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2019–2020

Harvey

2023

Mass Spectrometry Reviews

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This review is the tenth update of the original article published in 1999 on the application of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2020. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. The review is basically divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of arrays. (2) Applications to various structural types such as oligo‐ and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other areas such as medicine, industrial processes and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. The reported work shows increasing use of incorporation of new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented nearly 40 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show little sign of diminishing.

show abstract

A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase

Cited by 32 publications

References 24 publications

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

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

Cellular O‐Glycome Reporter/Amplification (CORA): Analytical and Preparative Tools to Study Mucin‐Type O‐Glycans of Living Cells

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2019–2020

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