Glucose unit index (GUI) of permethylated glycans for effective identification of glycans and glycan isomers

Gautam, Sakshi; Peng, Wenjing; Cho, Byeong Gwan; Huang, Yifan; Banazadeh, Alireza; Yu, Aiying; Dong, Xue; Mechref, Yehia

doi:10.1039/d0an00314j

Cited by 23 publications

(40 citation statements)

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“…Nevertheless, PGC-LC allows separation of permethylated GSL-glycan isomers. For future work, it might be intriguing to investigate GSL-glycan profiles of different cancer cell lines or biological fluids from patients of various diseases, as well as to assign glucose units for these glycans, as shown by Gautam et al [26]. The biggest pitfall of this technique may be the fact that the column used in this work is no longer produced by ThermoFisher.…”

Section: Discussionmentioning

confidence: 99%

“…For example, reduced native glycans can be analyzed using porous graphitized carbon (PGC) columns [24,25], while permethylated glycans are separated by C18 columns. This approach exploits the hydrophobicity of permethylated glycans [26] and therefore increases ionization efficiency, prompting higher sensitivity [27]. More recently, isomeric separation of permethylated N-glycans using PGC columns has been demonstrated by Zhou et al to preserve higher sensitivity facilitated by permethylation while delivering isomeric separation [28].…”

Section: Introductionmentioning

confidence: 99%

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Separation of Permethylated O-Glycans, Free Oligosaccharides, and Glycosphingolipid-Glycans Using Porous Graphitized Carbon (PGC) Column

2020

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Glycosylation is one of the most common and complex post-translational modifications of proteins. However, there are other carbohydrates such as free oligosaccharides and glycosphingolipids-glycans that are associated with important biological and clinical roles. To analyze these molecules using liquid chromatography coupled with mass spectrometry (LC-MS), the permethylation approach was utilized. Although permethylation is a commonly utilized glycan derivatization technique, separation of permethylated glycans released from glycosphingolipid (GSL) by LC-MS has never been previously demonstrated. Here, a nanoflow porous graphitized carbon (PGC) column coupled with a high-resolution mass spectrometer was used to achieve isomeric separation of these permethylated glycans. We demonstrate the separation of free reducing end and reduced end O-glycans, free oligosaccharides derived from human milk, and GSL glycans derived from the MDA-MB-231BR cancer cell line using PGC-LC-MS.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Separation of Permethylated O-Glycans, Free Oligosaccharides, and Glycosphingolipid-Glycans Using Porous Graphitized Carbon (PGC) Column

2020

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“…The data were obtained from the dextrin spiked human blood serum using LC-MS according to the previously published experimental protocols ( Gautam et al, 2020 ). Briefly, 1 μ g of dextrin was spiked as an internal standard in initial 10 μ l serum before permethylation.…”

Section: Methodsmentioning

confidence: 99%

“…The GUI approach was proposed for glycomics ( Campbell et al, 2008 ; Stockmann et al, 2013 ; Abrahams et al, 2018 ) and has been extensively used for the normalization of retention time of glycans ( Ashwood et al, 2020 ; Gautam et al, 2020 ; Fabini et al, 2001 ; Van den Steen et al, 2006 ; Royle et al, 2008 ). In our previous study, we examined the use of permethylated dextrin for the annotation of permethylated N-glycans and their isomers derived from standard glycoproteins and human blood serum ( Gautam et al, 2020 ). The calibrated GUI was proved to be reproducible across inter- and intra-laboratory analyses ( Gautam et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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GlycanGUI: Automated Glycan Annotation and Quantification Using Glucose Unit Index

et al. 2021

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The retention time provides critical information for glycan annotation and quantification from the Liquid Chromatography Mass Spectrometry (LC-MS) data. However, the variation of the precise retention time of glycans is highly dependent on the experimental conditions such as the specific separating columns, MS instruments and/or the buffer used. This variation hampers the exploitation of retention time for the glycan annotation from LC-MS data, especially when inter-laboratory data are compared. To incorporate the retention time of glycan across experiments, Glucose Unit Index (GUI) can be computed using the dextrin ladder as internal standard. The retention time of glycans are then calibrated with respect to glucose units derived from dextrin ladders. Despite the successful application of the GUI approach, the manual calibration process is quite tedious and often error prone. In this work, we present a standalone software tool GlycanGUI, with a graphic user interface to automatically carry out the GUI-based glycan annotation/quantification and subsequent data analysis. When tested on experimental data, GlycanGUI reported accurate GUI values compared with manual calibration, and thus is ready to be used for automated glycan annotation and quantification using GUI.

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Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

et al. 2021

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The wide variety of chemical properties and biological functions found in proteins is attained via post‐translational modifications like glycosylation. Covalently bonded to proteins, glycans play a critical role in cell activity. Complex structures with microheterogeneity, the glycan structures that are associated with proteins are difficult to analyze comprehensively. Recent advances in sample preparation methods, separation techniques, and MS have facilitated the quantitation and structural elucidation of glycans. This review focuses on highlighting advances in MS‐based techniques for glycomic analysis that occurred over the last 5 years (2017–2021) as an update to the previous review on the subject. The topics of discussion will include progress in glycomic workflow such as glycan release, purification, derivatization, and separation as well as the topics of ionization, tandem MS, and separation techniques that can be coupled with MS. Additionally, bioinformatics tools used for the analysis of glycans will be described.

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Glucose unit index (GUI) of permethylated glycans for effective identification of glycans and glycan isomers

Abstract: Retention time is the most common and widely used criterion to report the separation of glycans using Liquid Chromatography (LC), but it varies widely across different columns, instruments and laboratories....

Cited by 23 publications

References 50 publications

Separation of Permethylated O-Glycans, Free Oligosaccharides, and Glycosphingolipid-Glycans Using Porous Graphitized Carbon (PGC) Column

Separation of Permethylated O-Glycans, Free Oligosaccharides, and Glycosphingolipid-Glycans Using Porous Graphitized Carbon (PGC) Column

GlycanGUI: Automated Glycan Annotation and Quantification Using Glucose Unit Index

Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

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