Development of a 96-well plate sample preparation method for integrated<i>N</i>- and<i>O</i>-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry

Zhang, Tao; Madunić, Katarina; Holst, Stephanie; Zhang, Jing; Jin, Chunsheng; Dijke, Peter ten; Karlsson, Niclas G.; Stavenhagen, Kathrin; Wuhrer, Manfred

doi:10.1039/c9mo00180h

Cited by 60 publications

(94 citation statements)

References 44 publications

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“…O -glycan alditols released from RBD samples were prepared using a 96-well plate sample preparation method as previously described [22]. In brief, 20 μg of RBD samples were applied to the hydrophobic Immobilon-P PVDF membrane (Millipore, Amsterdam, The Netherlands) in a 96-well plate format.…”

Section: Methodsmentioning

confidence: 99%

“…Before desalting the collected samples were concentrated to approximately 30 μL under vacuum in a SpeedVac concentrator at 35°C for 2 h. Subsequently, 3 μL of glacial acetic acid were added to quench the reaction followed by brief centrifugation to collect the sample at the bottom of the well. Collected O -glycan alditols were then desalted and purified followed by a desalting and PGC clean-up using a 96-well plate based protocol [22]. Samples were dried in a SpeedVac concentrator directly in polymerase chain reaction (PCR) plates and redissolved in 10 μL of water prior to PGC nano-LC-ESI-MS/MS analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The analysis of O -glycan alditols was performed following a method described previously [22]. Measurements were performed on an Ultimate 3000 UHPLC system (Dionex/Thermo) equipped with an in house-packed PGC trap column (5 μm Hypercarb, 320 μm x 30 mm) and an in-house-packed PGC nano-column (Grace Discovery Sciences, Columbia, MD, USA) (3 μm Hypercarb 100 μm x 150 mm) coupled to an amaZon ETD speed ion trap (Bruker).…”

Section: Methodsmentioning

confidence: 99%

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Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

Gstoettner

Zhang

Resemann

et al. 2021

Preprint

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As the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

Gstoettner

Zhang

Resemann

et al. 2021

Preprint

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“…N-glycans of the CHO-Lec2 cells were analyzed as described previously ( 76 ). In short, cells were either harvested by trypsin treatment or alternatively scraped off, pelleted, washed, and stored until further use.…”

Section: Methodsmentioning

confidence: 99%

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

Szymczak-Kulus

Weidler

Bereznicka

et al. 2021

Journal of Biological Chemistry

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The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P k ) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli . A single amino acid substitution (p.Q211E) ramps up the enzyme’s promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

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“…The analyses of glycans using LC-MS can differ based on the derivatization techniques utilized for each experiment [23]. 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].…”

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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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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Development of a 96-well plate sample preparation method for integratedN- andO-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry

Abstract: The developed workflow allows high throughput sample preparation for glycomics analysis.

Cited by 60 publications

References 44 publications

Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

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

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