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Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells

  • Christoph Gstöttner
    Christoph Gstöttner
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
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  • , 
  • Tao Zhang
    Tao Zhang
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
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  • Anja Resemann
    Anja Resemann
    Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany
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  • Sophia Ruben
    Sophia Ruben
    InVivo BioTech Services GmbH, Neuendorfstr. 24A, 16761 Hennigsdorf, Germany
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  • Stuart Pengelley
    Stuart Pengelley
    Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany
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    Orcidhttp://orcid.org/0000-0003-2676-8096
  • , 
  • Detlev Suckau
    Detlev Suckau
    Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany
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    Orcidhttp://orcid.org/0000-0002-1859-2492
  • , 
  • Tim Welsink
    Tim Welsink
    InVivo BioTech Services GmbH, Neuendorfstr. 24A, 16761 Hennigsdorf, Germany
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  • Manfred Wuhrer
    Manfred Wuhrer
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
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    Orcidhttp://orcid.org/0000-0002-0814-4995
  • , and 
  • Elena Domínguez-Vega*
    Elena Domínguez-Vega
    Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
    *Email: [email protected]
    More by Elena Domínguez-Vega
    Orcidhttp://orcid.org/0000-0002-6394-0783
Cite this: Anal. Chem. 2021, 93, 17, 6839–6847
Publication Date (Web):April 19, 2021
https://doi.org/10.1021/acs.analchem.1c00893

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral 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 angiotensin-converting enzyme 2 (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 293 (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional post translational 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, a higher level of sialylation, and a combination of core 1 and core 2 type O-glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison.

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Since the outbreak of coronavirus disease 2019 (COVID-19), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has infected more than 100 million individuals and influences our daily lives. The coronavirus is an enveloped RNA virus containing three different structural proteins in the membrane, the envelop (E) protein, the membrane (M) protein, and the spike (S) glycoprotein. (1) The S protein is heavily glycosylated and forms a trimer on the SARS-CoV-2 surface. Each S protein carries 22 N-glycosylation sites (2) and consists of S1 and S2 subunits. Whereas the S2 subunit is necessary for membrane fusion, the S1 subunit directly interacts with the angiotensin-converting enzyme 2 (ACE2) receptor in the human respiratory tract and facilitates the entry into the host cell. (3) In particular, the receptor-binding domain (RBD) of the S1 subunit mediates the interaction with the ACE2 receptor. (4) This domain carries two N-linked glycans at positions N331 and N343 and, depending on the source, one or two O-linked glycosylation sites (T323/S325) are occupied. (2,5,6) The predicted O-glycosylation site at T323 is not present in the S protein of SARS-CoV-1 and has been hypothesized to have an influence on the interaction with the ACE2 receptor (7) and may be critical for conformational changes of the RBD. (8) Further studies have shown the role of glycosylation on ACE2–RBD binding. Crystallographic structures have confirmed the interaction between RBD and the ACE2 receptor and highlighted the importance of ACE2 glycosylation in the interaction. (9) However, the RBD used was expressed in insect cells, and the role of RBD glycosylation was not addressed. (9) In a more recent publication, the interaction of the N-glycan at position N343 of the RBD with the ACE2 receptor has been suggested by modeling the interaction using atomistic molecular dynamic simulations. (10) Another study revealed that the infectivity decreased by 1200 times (decrease of relative infectivity to 0.083%) when both N-glycosylation sites (N331 and N343) of the spike protein were silenced compared to the wild-type versions. These findings suggest that glycosylation is involved in the binding to the receptor, either directly or by providing conformational stabilization. (11) In line with its role in virus–ACE2 interaction, the RBD is the primary target of neutralizing antibodies. (12) Interestingly, an antibody named S309, pulled from serum from a SARS-CoV-1 recovered patient, bound an epitope containing also glycosylation. (13) This binding site is also conserved in SARS-CoV-2 and was predicted as an epitope for neutralizing antibodies. (14) The neutralizing S309 antibody was shown to bind to a protein epitope (331–344) in combination with the glycan at N343. The antibody showed especially strong interaction with the core fucose and to a minor extent with the remaining glycan structure. (13) All of these findings emphasize that the assessment of RBD glycosylation is highly important.
Recombinantly produced S proteins are essential tools in the fight against SARS-CoV-2, contributing to a further understanding of the interaction mechanism, providing efficient components for diagnostic purposes and helping in vaccine development. (15) However, it is important to understand that glycosylation and other structural characteristics may differ considerably between different biotechnologically produced proteins and their natural forms. Considering the relevance of RBD glycosylation on ACE2 binding and recognition by neutralizing antibodies, the use of well-characterized S proteins is essential. The S protein, in particular, has been produced as a full-length protein as well as in a short version containing the RBD. (16) Site-specific glycosylation analysis of the 22 N-glycosylation sites of the recombinant S glycoprotein expressed in human embryonic kidney 293 (HEK293) cells showed mainly complex type glycans but also, at certain glycosylation sites, high mannose structures and in lower amounts, some hybrid structures. (2,5) In particular, the two N-glycosylation sites (N331 and N343), which are located in the RBD were found to carry mainly complex type glycans as well as spurious amounts of high mannose glycans. Similarly, for the S1 subunit recombinantly produced in HEK293 cells, mainly complex type glycans were found at both sites. (5) As predicted by Uslupehlivan and Şener, (7) O-glycosylation at the position T323 and/or at S325 was found. Whereas one report, analyzing the whole S protein recombinantly produced, showed only trace levels of O-glycans, (2) another study detected high levels of O-glycosylation. (6) Still, in these studies, the localization of the O-glycosylation site at T323 and/or S325 was not possible. O-glycosylation at these two positions is also thought to be important to stabilize the conformation of the RBD or to introduce conformational changes. (8) All of these studies have been performed using recombinant versions of the spike protein or subunits thereof. Interestingly, upon expression in HEK293 cells, the amount of sialic acids varied from low to high depending on whether the complete S protein or only the S1 subunit was produced, (5) suggesting that the N- and O-glycosylation is dependent on the context of the protein (S, S1, or RBD only). So far, only terminal glycan epitopes of HEK293-produced RBD have been studied by NMR; however, an assessment of the entire N-glycan structure, composition, and the relative quantification of the N-glycans could not be achieved. (17) Furthermore, the O-glycosylation was neglected completely. Also, a characterization of the intact RBD is still missing, providing information on the combination of the glycans as well as on additional protein backbone modifications.
Here, we present an in-depth structural and functional characterization of two commercially available SARS-CoV-2 RBDs produced in two different expression systems, HEK293 and Chinese hamster ovary (CHO) cells. To achieve comprehensive structural information, a multilevel characterization was performed (Figure 1). Both RBD samples were initially analyzed at the intact level by top-down sequencing using matrix-assisted laser desorption ionization (MALDI) in-source-decay (MALDI-ISD) mass spectrometry (MS) (18,19) and by sheathless capillary electrophoresis (CE)-MS after full N- and O-deglycosylation to establish their protein sequences and putative nonglycan modifications. With the established sequences and the help of sequential glycosidase treatment, the glycoforms on both N- and the O-glycosylation sites were assigned. Our findings were confirmed by glycopeptide analysis and the analysis of released O-glycans by porous graphitized carbon (PGC) nanoliquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). To assess functional differences between the two RBD samples, we determined their binding characteristics to ACE2 and sera of patients who recovered from a previous SARS-CoV-2 infection.

Figure 1

Figure 1. Multilevel characterization of CHO- and HEK293-RBD. Next to intact protein analysis using CE-MS and MALDI-ISD-MS, the samples are structurally characterized by glycan dissection, glycopeptide analysis, and released glycan analysis. Their functional characterization was performed by measuring their binding characteristics to ACE2 and anti-SARS-CoV-2 antibodies.

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Experimental Section

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Reagents and Samples

Reagents used for this study were at least of analytical grade; for more details, see Supporting Information S1. Recombinant RBDs (Wuhan-Hu-1-isolate (MN908947)), either transiently expressed in HEK293 or stably expressed in CHO cells, were used (InVivo Biotech Services, Henningsdorf, Germany). The constructs contained the amino-acid sequence 319 to 541 with a C-terminal 6xHis-Tag. Recombinant RBDs were purified using immobilized metal affinity chromatography and a size-exclusion polishing step. The samples were stored in 20 mM sodium phosphate, 300 mM NaCl, pH 7.2. Glycosidases SialEXO (sialidases α2-3, α2-6, and α2-8), GalactEXO (galactosidases β1-3 and β1-4), OglyZOR (endo-α-N-acetylgalactosaminidase), OpeRATOR (O-protease), α1-2 fucosidase, and α1-3,4 fucosidase were obtained from Genovis (Lund, Sweden). Peptide N-glycosidase F (PNGaseF) was purchased from Roche Diagnostics (Mannheim, Germany).

Intact RBD Analysis by Sheathless CE-MS

Samples for intact protein analysis were buffer exchanged with Tris pH 6.8 using 10 kDa Vivaspin MWCO filters Sartorius (Göttingen, Germany) to a final concentration of 1 μg/μL. Subsequently the enzymes SialEXO, GalactEXO, OglyZOR, or OpeRATOR were added according to the producer specifications and incubated overnight at 37 °C. PNGaseF was added in a ratio of 1:5 (v/v) and incubated overnight at 37 °C. For removing antenna fucosylation, 20 μg of the HEK293-RBD sample was incubated with 4.4 μg of fucosidase α1-2 or 8.8 μg of fucosidase α1-3,4 overnight at 37 °C. Afterward, all samples were buffer exchanged with 100 mM ammonium acetate, pH 3.0, and subsequently analyzed by sheathless CE-MS. For sheathless CE measurements, a Sciex CESI 8000 instrument combined with a Bruker Impact qTOF-MS was used. Bare-fused silica CE capillaries were obtained from Sciex (Framingham, MA) and subsequently coated with polyethylenimine (PEI). (20) Afterward, the sample was injected (15 s, 5 psi) and analyzed by applying 20 kV (reversed polarity) at 20 °C for 45 min. For detailed CE and MS settings, see Supporting Information S2. Intact RBD data were deconvoluted using DataAnalysis 5.3 (Bruker Daltonics) maximum entropy algorithm. Deconvoluted mass spectra were baseline subtracted and smoothed using the Gaussian smoothing function with a width of 0.5 Da and one cycle. Glycoforms were assigned based on the average masses observed and observed mass shifts with enzyme treatments. Reconstructed intact spectra from bottom-up data were simulated using an algorithm in R developed by Yang et al., which is publicly available and can be found at https://github.com/Yang0014/glycoNativeMS. (21) For our data, instead of simulating the charge-state distribution of the protein, we simulated the deconvoluted mass spectra so no correction for the relative abundance of different charge states needed to be performed.

MALDI-ISD Top-Down Protein Sequence Analysis

Fully deglycosylated RBD samples were reduced for 30 min at 50 °C using dithiothreitol (DTT). Two microliters of the reduced sample was spotted on a hydrophilic anchor of an MTP BigAnchor sample plate and incubated. After 2 min, the remaining droplet was removed and the spot was washed using 0.1% trifluoroacetyl (TFA) in water. Subsequently, 1 μL of a super-DHB (sDHB) matrix solution (25 μg/μL in 50% acetonitrile/49.9% water/0.1% TFA) was deposited on the dried sample spot. MALDI-ISD spectra were acquired with a rapifleX MALDI-TOF MS instrument (Bruker). Detailed MS settings and spectra assignment can be found in Supporting Information S3.

Glycopeptide Analysis by Reverse-Phase Liquid Chromatography (RPLC)-MS/MS

For glycopeptide generation, double digestion with trypsin followed by elastase was performed. In short, samples were reduced (45 mM DTT), alkylated (100 mM iodoacetamide (IAA)), and the reaction was stopped (45 mM DTT). After overnight tryptic digestion, the reaction was stopped (90 °C) and elastase was added. After overnight incubation with elastase, the reaction was stopped (formic acid). A more detailed description can be found in Supporting Information S4. The digested samples were separated using a nanoElute (Bruker) nanoflow ultrahigh-performance liquid chromatography (UHPLC) equipped with an Aurora 25 cm × 75 μm C18 column with a particle size of 1.6 μm (IonOpticks, Parkville, Victoria, Canada) and analyzed on a timsTOF PRO (Bruker). Information about the LC and MS parameters can be found in Supporting Information S5. MS/MS data of N-linked glycopeptides were generated following Hinneburg et al. (22) and processed using DataAnalysis 5.3 and MGF peak lists were imported into a BioPharma Compass 2021 (Bruker) and further analyzed using the glycopeptide analysis workflow. This workflow includes MS/MS spectra classification using typical fragmentation patterns to determine the peptide mass and the glycan mass. The peptide sequences were then identified using the theoretical digest feature of the software. In a second search, the classified MS/MS spectra were analyzed using the GlycoQuest search engine within BioPharma Compass 2021 software. For quantification, the most intense isotope of each charge state ([M + H]+, [M + 2H]2+, or [M + 3H]3+) was extracted and the results normalized to the total peak intensity of all glycopeptides within one sample to 100%.

Released O-Glycan Analysis by PGC Nano-LC-ESI-MS/MS

Released O-glycan alditols from RBD samples were prepared using a 96-well plate sample preparation method, as previously described. (23) In brief, after N-glycan removal (PNGaseF, 2 U, overnight incubation at 37 °C), the O-glycans were released via reductive β-elimination. The analysis of O-glycan alditols was 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 nanocolumn (Grace Discovery Sciences, Columbia, MD) (3 μm Hypercarb 100 μm x 150 mm) coupled to an amaZon ETD speed ion trap (Bruker) following a method described previously. (23) More detailed information can be found in the Supporting Information (Section S6). Structures of detected glycans were confirmed by MS/MS in a negative mode. (24) Glycan structures were assigned on the basis of the known MS/MS fragmentation patterns in a negative-ion mode, (25−27) elution order, and general glycobiological knowledge, with help of Glycoworkbench (28) and Glycomod software. (29) Relative quantification of individual glycans was performed by normalizing the total peak area of all glycans within one sample to 100%.

SARS-CoV-2-IgG Enzyme-Linked Immunosorbent Assay (ELISA) with RBD Antigens

The antigens (intact or glycosidase-treated HEK293-RBD or CHO-RBD) were used to coat immunoassay plates at a concentration of 2 μg/mL. Subsequently, blocking was performed with blocking buffer containing 1% bovine serum albumin (BSA). Serum collected >10 weeks after the onset of first symptoms from 12 SARS-CoV-2 polymerase chain reaction (PCR)-positive tested donors was applied to the RBD-coated wells at a dilution of 1:101. As a negative control, a 1:101 diluted serum pool containing 10 sera taken from healthy individuals in the year 2012 was used. The wells were washed three times with 250 μL of phosphate-buffered saline (PBS) containing 0.1% Tween 20. Subsequently, bound IgG from sera was detected by anti-human IgG-horseradish peroxidase (HRP) and developed with tetramethylbenzidine. The reaction was stopped using sulfuric acid. The absorbance was measured at 450 nm using a microplate reader. To determine the linear correlation between the absorption values, the Pearson correlation was calculated using GraphPad Prism 9. Linear regression was employed for the visualization of the correlation. For this analysis, the negative control value was excluded.

ACE2 Receptor-Binding Assay

Intact and glycosidase-treated RBD, S1 subunit, and the S protein (InVivo Biotech) were biotinylated using 10 molar excess of NHS-LC-Biotin (Thermo Scientific). Immunoassay plates were coated with 2.5 μg/mL recombinant angiotensin-converting enzyme 2 (ACE2, Sigma-Aldrich). Subsequently, the assay plate wells were blocked using blocking buffer containing 1% BSA. Dilutions of the biotinylated antigens ranging from 1 to 0.001 μg/mL were applied to the ACE2-coated assay plate wells in duplicates. The wells were washed three times with 250 μL of PBS containing 0.1% Tween 20. Bound biotinylated antigens were detected using a streptavidin peroxidase conjugate (Roche) and developed with tetramethylbenzidine. The reaction was stopped using sulfuric acid. The absorbance was measured at 450 nm using a microplate reader. GraphPad Prism 9 was used to plot log(dose) response curves (variable slope, four parameters) and to compute nonlinear fits, which were utilized to calculate the half-maximal concentrations (EC50).

Results and Discussion

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Structural Characterization of CHO- and HEK293-RBD

We characterized the RBD domains (amino acids 319-541 containing a C-terminal His-tag) produced in CHO and HEK293 cells. This domain contains two N-glycosylation sites at positions N331 and N343 as well as two potential O-glycosylation sites at T323 and S325. Analysis of the intact RBDs revealed a complex pattern of signals comprising different N- and O-glycans and additional protein backbone modifications (see the Assessment of RBD N-glycosylation section). Therefore, to unravel this heterogeneity and achieve comprehensive structural characterization, we used a multilevel approach (Figure 1). Next, to classical released glycan and glycopeptide approaches, we applied a step-by-step dissection of glycans at the intact level similar to the work of Wohlschlager et al. for the biopharmaceutical etanercept. (30)

Characterization of the Protein Backbone after N- and O-Glycan Removal

To get information on the integrity of the protein backbone, the RBD proteins were treated with PNGaseF to remove the N-glycans and with an endo-α-N-acetylgalactosaminidase in combination with a mix of sialidases to remove the O-glycans. After deglycosylation, the RBDs were analyzed by CE-MS for intact mass and MALDI-ISD MS for top-down sequencing. Analysis of the samples by CE-MS resulted in one main peak in the base peak electropherogram (BPE) corresponding to the RBD. For the RBD produced in CHO cells, the observed averaged mass (26033.9 Da) was 119.0 Da higher than the theoretical mass calculated solely based on the amino-acid sequence (25914.9 Da) (Figure 2A). As the RBD contains a free cysteine at position C538, (31) it was presumably cysteinylated (+119.1 Da). This modification is often observed for free light chains during antibody production using CHO cells. (32) After reducing the disulfide bonds with DTT, the mass of the deglycosylated and completely reduced protein was 25923.0 Da, which is consistent with the expected theoretical mass (25923.0 Da), confirming the presence of cysteinylation (data not shown). For the RBD expressed in HEK293 cells, a deconvoluted mass of 26144.9 Da was observed (Figure 2B). Considering a cysteinylation of the free cysteine also in the HEK293-RBD, an additional mass difference of 110.9 Da was observed compared to the theoretical mass indicating additional modifications.

Figure 2

Figure 2. Deconvoluted ESI mass spectra of the main peak observed after sheathless CE-MS for (A) CHO-RBD and (B) HEK293-RBD. HEK293-RBD carries an additional N-terminal pyroglutamate. Blue square, N-acetylglucosamine; yellow square, N-acetylgalactosamine; and yellow circle, galactose.

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Both RBD samples were analyzed at the intact level by top-down sequencing using MALDI-ISD MS after full reduction and N- and O-deglycosylation. MALDI-ISD provides large terminal sequence tags and detects terminal (and internal) post translational modification (PTMs) from intact proteins. For both RBDs, we detected a C-terminal sequence tag of 73 amino acids (root-mean-square (RMS) error of 0.028 Da) verifying the expected C-terminus of the sequence. The N-terminal sequence tag, however, was not consistent between both RBDs. While the CHO-RBD sequence was in full agreement with the MALDI-ISD spectrum (Figure S1A), the HEK293-RBD sequence was found to be N-terminally extended by pyro-Glu (mass shift of 111.03 Da). Overall, 52% of the sequence was confirmed this way including an N-terminal sequence tag of 46 residues for the nonglycosylated and 29 residues for the O-glycosylated form (Figure S1B). Of note, the HEK293-RBD came with a slightly different sequence compared to the CHO-RBD, resulting in an additional glutamine at the N-terminus after cleavage of the signaling peptide (Figure S1B). Taking pyroglutamic acid formation into consideration, the measured mass perfectly fits the theoretical mass (26145.1 Da). The N-terminal pyroglutamic acid formation was further confirmed by bottom-up analysis (data not shown). In addition, we found a species migrating before the main signal in the BPE with a +42.3 Da mass difference (26187.4 Da). This difference in mass might correspond to acetylation, however, it could not be confirmed by bottom-up analysis after trypsin digestion. This species was only observed in HEK293-RBD and not in CHO-RBD.

RBD O-Glycan Characterization

The RBD has two potential O-glycosylation sites at position T323 and S325. To characterize the O-glycans, the de-N-glycosylated RBDs (after treatment with PNGaseF) were analyzed by CE-MS (Figure S2) and the released O-glycans by PGC nano-LC-ESI-MS/MS (Figure 3), respectively. Table S1 summarizes the obtained results. In CHO-RBD, mainly core 1 structures with two sialic acids (H1N1S2) were observed. HEK293-RBD showed a much more diverse O-glycosylation pattern with a core 1 structure with two sialic acids as the main signal. Additionally, several core 2 structures, with and without fucose as well as with sulfation were detected. These data are in accordance with the data in Figure 2B for the sample treated with endo-α-N-acetylgalactosaminidase, where an additional signal of 26875.7 Da was observed. This mass corresponds to an H2N2 modification (theoretical mass 26875.8 Da), which is presumably a core 2 O-glycan, which cannot be cleaved by the endo-α-N-acetylgalactosaminidase. Similar glycoforms were not detected in CHO-RBD. The position of fucoses, either to the terminal galactose or the N-acetylglucosamine were confirmed by MS fragmentation and treatment with different fucosidases (Figure S3).

Figure 3

Figure 3. PGC nano-LC-ESI-MS/MS of released O-glycans from (A) CHO-RBD and (B) HEK293-RBD. Yellow square, N-acetylgalactosamine; yellow circle, galactose; blue square, N-acetylglucosamine; red triangle, fucose; purple diamond, N-acetylneuraminic acid (sialic acid); and S, sulfate.

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Hitherto published S protein bottom-up studies have not revealed the O-glycan attachment site, leaving both T323 and S325 as valid options. (2,6) To resolve this, we follow a different strategy based on N-glycan removal (with PNGaseF) and O-protease cleavage using the enzyme OpeRATOR in combination with a mix of sialidases. The O-protease cleaves the protein at the N-terminal site of an O-glycosylation site (Figure S3). This would result in a loss of the amino acids 319–322 from the RBD if the O-glycosylation is on T323 or 319–324 in case S325 carries O-glycan. As shown in Figure S4A, only one signal with a mass of 25919.0 Da was observed for the CHO material, which correlates to the RBD cleaved at T323 with a core 1 O-glycan H1N1 (theoretical mass 25918.8 Da). No RBD cleaved at S325 was observed. In HEK293-RBD, the same signal with a core 1 glycan H1N1 was observed also indicating that the glycosylation is located at T323 (Figure S4B). Next to this signal, an amount of uncleaved RBD with presumably core 2 O-glycan structures was detected. This is in line with a recent article that shows that the used O-protease can cleave N-terminal to core 1 but not core 2 glycan structures, similar to endo-α-N-acetylgalactosaminidase. (33) To confirm that the core 2 structures are located at T323, we performed MALDI-ISD MS analysis using super-DHB as a matrix. In contrast to collision-induced dissociation (CID) fragmentation in bottom-up analysis, MALDI-ISD is known to result in mainly singly charged c– and z + 2 ions with labile modifications remaining intact, which allows localizing O-linked glycosylation sites. MALDI-ISD MS of CHO-RBD after N-glycan and sialic acid removal showed N-terminal fragments (c-ions) with core 1 structure (H1N1) for the site T323 and no additional glycosylated fragments at S325 (+365.1 Da) could be detected, confirming the presence of the O-glycosylation at T323 (Figure S5A). No fragments of T323 without glycosylation were observed, indicating full-site occupancy. The analysis of HEK293-RBD comprising only core 2 structure O-glycans (after N-glycan and core 1 O-glycan removal) clearly showed that the core 2 structures are also located on T323 (Figure S5B). In conclusion, the combination of intact analysis of O-protease-treated RBDs together with MALDI-ISD allowed localizing of the O-glycans to the T323 with high confidence.

Assessment of RBD N-Glycosylation

We studied the two N-glycosylation sites N331 and N343 using a bottom-up glycopeptide approach combined with a step-by-step dissection of glycans at the intact level. (30) Whereas the glycopeptide data yielded the glycan composition per site, we studied the combination of these glycans at the intact level. (21)
The intact RBDs were first incubated with a mixture of sialidases and galactosidases. These enzymes removed the sialic acids and the terminal galactoses on the N- as well as O-glycans, resulting in considerably simplified deconvoluted mass spectra, as shown in Figures S6 and S7. Overall, mainly fucosylated complex type glycans were observed for both RBDs (>90.0%). This is in line with previous studies on HEK293-produced intact S protein or S1 subunit. (2,5) The dissection of the terminal galactoses also permits a direct distinction between N-acetyllactosamine (LacNAc) repeats and additional antenna. CHO-RBD showed a higher relative abundance of LacNAc repeats than HEK293-RBD. Additionally, CHO-RBD showed a higher antennarity, with two triantennary structures as the most abundant glycoforms, contrary to HEK293 material with di- and triantennary glycans as major signals. This was confirmed by the analysis of the glycopeptides (Figure S8, Tables S2, and S3). In general, more di- and triantennary structures were observed for HEK293-RBD (N331: 78.4% and N343: 84.5%) compared to the CHO-RBD (N331: 48.1% and N343: 72.1%). Therefore, CHO-RBD showed a larger contribution of tetraantennary structures and LacNAc repeats (N331: 42.7% and N343: 24.4%) compared to HEK293-RBD (N331: 15.7% and N343: 9.9%). Between both glycosylation sites. a difference in the number of LacNAc repeats and high antennary structures was observed with minor amounts on N343 compared to N331.
In addition, we found spurious amounts of hybrid-type glycans only at N343 and only in HEK293-RBD (0.8%), similar to previous publications. (2,5) Regarding high mannose glycans, low amounts were detected for both RBDs. In the case of HEK293-RBD, Man5 and Man6 glycans with and without phosphorylation were observed, whereas in the case of CHO-RBD, Man5 and Man6 glycans carrying an additional phosphate were detected. For HEK293-RBD, the high mannose and phosphorylated high mannose structures show combined abundances of 3.1% (N331) and 1.3% (N343) in line with the findings of Watanabe et al. (2) for the full-length S protein. For CHO-RBD, the distribution was more skewed with 7.7% (N331) and 0.9% (N343).
Besides, in the deconvoluted mass spectrum of HEK293-RBD treated with sialidase and galactosidase a pattern of signals with a mass shift of +308.1 Da was observed. A combination of one galactose and one fucose could explain this mass shift. It was shown in the literature that β-galactosidases are not able to remove the terminal galactose if antenna fucose, either linked to the galactose itself or to the N-acetylglucosamine, is present. (34) Therefore, we incubated HEK293-RBD either with α1-2 fucosidase and in parallel with α1-3,4 fucosidase to remove fucoses linked to the galactose and N-acetylglucosamine, respectively. Incubation with these fucosidases allowed the β-galactosidase to remove the terminal galactose. As shown in Figure S3, after removing the differently linked antenna fucoses, the +308.1 signals disappear completely confirming antenna fucosylation and providing information on the linkage of the antenna fucose with a predominant linkage to the N-acetylglucosamine. Additionally, antenna fucosylation in HEK293-RBD was confirmed by glycopeptide analysis (26.7 and 33.8% of antenna fucosylation on N331 and N343, respectively) (Figure S9, Tables S2, and S3). No antenna fucosylation was found for CHO-RBD with any of the approaches.
The analysis of the intact RBDs without any previous enzymatic treatment resulted in a very complex mass spectrum (Figures 4 and S10).

Figure 4

Figure 4. Deconvoluted mass spectra of the intact RBD (not enzymatically treated) produced by HEK293 cells. The assignments were based on previous enzyme treatments, mass, and glycopeptide as well as released O-glycan data. Peaks marked with an asterisk * are presumably the acetylated variant of RBD. Yellow square, N-acetylgalactosamine; yellow circle, galactose; blue square, N-acetylglucosamine; red triangle, fucose; and purple diamond, N-acetylneuraminic acid (sialic acid).

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Based on the information obtained for the released O-glycans, glycopeptide, and enzymatically treated intact RBDs, the spectra were confidently assigned. In particular, the HEK293-RBD exhibited a large heterogeneity. In addition to the variability resulting from the acetylation and antenna fucosylation, which were not observed in CHO-RBD, also a higher degree of sialylation was observed for HEK293-RBD. This was also supported by the glycopeptide data in which CHO-RBD showed only 2.5 or 0.8% sialylated glycans, whereas HEK293-RBD contained 56.4 or 36.3% on N331 or N343, respectively (Figure S11, Tables S2, and S3). Furthermore, in CHO-RBD, only monosialylated species were observed, while in the HEK293-RBD, mono-, di-, or trisialylated species were detected. Interestingly, these high sialylation levels were not observed by Watanabe et al. who reported only 22% sialylation on N331 and 4% sialylation on N343 for the full-length S protein. (2) This might be due to the different constructs with RBD expression vs the S1 subunit or S protein. A similar effect of lower sialylation on the complete S protein has also been previously reported by comparing the entire S protein expressed in HEK293 cells with the S1 subunit. (5) Whereas the S protein carried either ∼40 or 10% sialylation on N331 or N343, the S1 subunit carried 80 or 50% on N331 or N343, respectively. These findings highlight that sialylation, which influences the isoelectric point of a protein, can change with the length of the protein expressed (S, S1, or RBD) and must be taken into account.
Finally, to support our assignments, the intact mass spectra were reconstructed from the glycopeptide data as described. (21)Figure 5A shows a very strong correlation for CHO-RBD, confirming the assignments. For HEK293-RBD, the reconstructed mass spectrum showed a shift toward lower masses compared to the intact profile (Figure 5B). Proteins with higher sialylation levels often show a discrepancy between the intact and the glycopeptide-reconstructed mass spectra, (21) which could be attributable to a nonrandom combination or to a biased ionization efficiency of sialylated glycopeptides.

Figure 5

Figure 5. Deconvoluted mass spectra of the RBD intact (nonenzymatical treated) (black trace) and in silico simulated mass spectrum (vermillion trace) of RBD produced in (A) CHO cells or (B) HEK293 cells.

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Functional Characterization of CHO- or HEK293-RBD

Next to the structural characterization of the RBD, a functional characterization was also performed. To assess the RBD functionality, an ACE2 receptor-binding assay and a binding assay for anti-RBD antibodies from COVID-19 patient sera were performed.

SARS-CoV-2 Antibody Binding Assay

The binding of anti-SARS-CoV-2-IgG antibodies in sera taken from 12 COVID-19 patients more than 10 weeks after the onset of the first symptoms and RBD was determined using an ELISA assay. For the assay, the intact CHO- or HEK293-RBD, as well as deglycosylated RBD samples, were used. As a negative control, a pool of 10 sera collected in 2012 was used. As expected, higher absorption values were observed for the 12 COVID-19 patient sera compared to the negative control, indicating binding of specific antibodies to the RBDs (Figure S12). Both intact RBD samples showed similar absorption values, while absorption values were slightly elevated when the deglycosylated RBDs were employed. Also, for the negative controls, the deglycosylated RBD showed elevated absorption values compared to the intact RBD. This is most probably the result of unspecific binding, which may be increased after deglycosylation. This was also reflected in the correlation of the absorption values of the patient sera. The correlation of these values gained using intact RBDs and their deglycosylated version (CHO: R2 09681, HEK293: 0.9755) was slightly lower than the correlation obtained using intact RBD produced by CHO and HEK293 cells (R2 0.9894) or deglycosylated CHO- vs deglycosylated HEK293-RBD (R2 0.9905) (Figure S13).

ACE2 Receptor-Binding Assay

Using a plate-based ACE2 receptor-binding assay, dose-dependent binding of both CHO- and HEK293-RBD was observed (Figure 6A). Comparing the RBDs expressed in both production systems, similar binding properties were observed with a trend toward a lower EC50 value for CHO-RBD. As glycosylation has been hypothesized to have a role in the interaction with ACE2, (10,11) we additionally tested the RBDs after deglycosylation. Deglycosylation was found to reduce the binding of the RBDs, as reflected in an approximately 2 times increase of the EC50 values. Of note, Qianqian et al. reported that the infectivity is reduced to only 0.08% after removal of the N-glycosylation sites. (11) Our results, although they show a slight variation between the glycosylated and nonglycosylated versions, do not explain this drastic difference in infectivity. Furthermore, the presence of endoglycosidases could also have affected the biotinylation rate, resulting in the overall reduced binding values. Supporting the hypothesis of Qianqian, these glycans may be crucial for stabilizing the trimeric spike protein rather than influencing the binding affinity. (11)

Figure 6

Figure 6. ACE2 binding assay. Dose–response curves of CHO- or HEK293-RBD binding to the ACE2 receptor. Comparison to (A) deglycosylated version of CHO- or HEK293-RBD and (B) S or S1 subunit (produced in HEK293 cells).

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Additionally, we compared the ACE2 receptor-binding affinity of both RBDs to the S1 subunit, as well as the intact S protein. The binding affinity of the ACE2 receptor to the RBDs was significantly increased compared to the S1 subunit and the S protein (Figure 6B). This might be due to the higher accessibility to ACE2 of the RBD only compared to the S1 subunit or even the S protein. As shown by Casalino et al., in the trimeric S protein, the RBD can be in an up or down conformation and, therefore, more or less accessible to the ACE2 binding. (8) Using the S protein or the S1 subunit, some parts of the RBD might not be accessible or less accessible for the ACE2 receptor, which may explain the reduced binding affinity. These results highlight the importance of the proper selection of recombinant proteins.

Conclusions

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RBD proteoforms were comprehensively characterized by combining intact protein, glycopeptide, and released glycan analyses with enzymatic glycan dissection and top-down sequencing. The combination of multiple MS workflows was fundamental for assigning the intact RBD proteoforms. In particular, glycan dissection of the intact protein using sequentially different glycosidases has shown to be very powerful to annotate complex intact RBD spectra. The glycopeptide data, next to providing site-specific information, were used to simulate an in silico intact spectrum and corroborate our assignments. This approach was applied to RBD samples from both CHO and HEK293 cells. In the case of the low sialylated CHO-RBD, a very strong correlation was obtained. However, for HEK293-RBD, the simulated spectrum showed a clear shift to a lower mass associated with the loss of sialic acids or an ionization bias of the glycopeptides. This stresses the importance of assessing intact proteoforms to avoid skewing the data in any direction. The observed differences in N-glycosylation, with higher sialylation levels and antenna fucosylation for HEK293-RBD and low sialylation levels but high antennary and LacNAc repeat structures for CHO-RBD are typical for the two expression systems. For O-glycans, CHO-RBD showed mainly core 1 type glycans while HEK293-RBD presented a combination of core 1 and core 2 type O-glycans. Furthermore, using alternative approaches, such as N-terminal cleavage at the O-glycosylation site and MALDI-ISD, we localized the O-glycosylation site to T323, previously unknown. Further steps will focus on validation of the method for batch-to-batch comparison of different RBD batches and for release testing in companies producing RBD-based vaccines. Full structural characterization of the S protein instead of RBD would be challenging due to the higher molecular mass and heterogeneity (22 N-glycosylation sites). Still, some of the strategies applied in this approach, as complete deglycosylation and analysis of protein backbone or N-terminal cleavage at the O-glycosylation sites, could provide additional information to previously published studies. From a functional point of view, both RBDs showed similar binding to antibodies from COVID-19 patient sera as well as to the ACE2 receptor. The ACE2 binding was increased compared to the S protein or S1 subunit, likely due to higher accessibility. After deglycosylation, the binding of RBDs to antispike antibodies remained unaffected. For ACE2, a minor decrease in the EC50 values was observed, which does not fully explain the infectivity decrease with aglycosylation observed in previous studies. These findings suggest that glycosylation of the RBD plays a role in conformational stabilization rather than affecting binding affinity between ACE2 and RBD. Further studies are warranted on the influence of RBD glycosylation on S conformation, ACE2 binding, as well as virus infectivity and biology.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.1c00893.

  • Details on the Experimental Section; MALDI-ISD sequence coverage; deconvoluted mass spectra for the de-N-glycosylated, fucosidase-treated, and O-protease-treated intact RBDs; O-glycan site localization by MALDI-ISD; deconvoluted mass spectra for the galactosidase- and sialidase-treated RBDs; site-specific glycan-type distribution pie charts; results for the SARS-CoV-2 antibody binding assay; and relative quantification of O-released glycans and glycopeptides from RBDs (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Christoph Gstöttner - Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
    • Tao Zhang - Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
    • Anja Resemann - Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany
    • Sophia Ruben - InVivo BioTech Services GmbH, Neuendorfstr. 24A, 16761 Hennigsdorf, Germany
    • Stuart Pengelley - Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;  Orcidhttp://orcid.org/0000-0003-2676-8096
    • Detlev Suckau - Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany;  Orcidhttp://orcid.org/0000-0002-1859-2492
    • Tim Welsink - InVivo BioTech Services GmbH, Neuendorfstr. 24A, 16761 Hennigsdorf, Germany
    • Manfred Wuhrer - Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands;  Orcidhttp://orcid.org/0000-0002-0814-4995
  • Notes
    The authors declare the following competing financial interest(s): InVivo BioTech Services is a biotechnology company producing antibodies and proteins, including SARS-CoV-2 antigens.

Acknowledgments

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The authors thank Eckhard Belau and Waltraud Evers for preparing the samples and analysis. This work was supported by the Analytics for Biologics project (Grant Agreement ID 765502) of the European Commission.

References

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This article references 34 other publications.

  1. 1
    Li, H.; Liu, S.-M.; Yu, X.-H.; Tang, S.-L.; Tang, C.-K. Coronavirus disease 2019 (COVID-19): current status and future perspectives. Int. J. Antimicrob. Agents 2020, 55, 105951  DOI: 10.1016/j.ijantimicag.2020.105951
    Google Scholar
    1
    Coronavirus disease 2019 (COVID-19): current status and future perspectives
    Li, Heng; Liu, Shang-Ming; Yu, Xiao-Hua; Tang, Shi-Lin; Tang, Chao-Ke
    International Journal of Antimicrobial Agents (2020), 55 (5), 105951CODEN: IAAGEA; ISSN:0924-8579. (Elsevier B.V.)
    A review. Coronavirus disease 2019 (COVID-19) originated in the city of Wuhan, Hubei Province, Central China, and has spread quickly to 72 countries to date. COVID-19 is caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [previously provisionally known as 2019 novel coronavirus (2019-nCoV)]. At present, the newly identified SARS-CoV-2 has caused a large no. of deaths with tens of thousands of confirmed cases worldwide, posing a serious threat to public health. However, there are no clin. approved vaccines or specific therapeutic drugs available for COVID-19. Intensive research on the newly emerged SARS-CoV-2 is urgently needed to elucidate the pathogenic mechanisms and epidemiol. characteristics and to identify potential drug targets, which will contribute to the development of effective prevention and treatment strategies. Hence, this review will focus on recent progress regarding the structure of SARS-CoV-2 and the characteristics of COVID-19, such as the etiol., pathogenesis and epidemiol. characteristics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmvFSksLs%253D&md5=c4a9e77b6383b16414b6969186015854
  2. 2
    Watanabe, Y.; Allen, J. D.; Wrapp, D.; McLellan, J. S.; Crispin, M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science 2020, 369, 330– 333,  DOI: 10.1126/science.abb9983
    Google Scholar
    2
    Site-specific glycan analysis of the SARS-CoV-2 spike
    Watanabe, Yasunori; Allen, Joel D.; Wrapp, Daniel; McLellan, Jason S.; Crispin, Max
    Science (Washington, DC, United States) (2020), 369 (6501), 330-333CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This anal. enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiol. and vaccine design.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVSjtLnE&md5=e751e60b601f2c20ff3f1b1d0ef45a3f
  3. 3
    Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T. S.; Herrler, G.; Wu, N.-H.; Nitsche, A.; Müller, M. A.; Drosten, C.; Pöhlmann, S. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020, 181, 271– 280.e8,  DOI: 10.1016/j.cell.2020.02.052
    Google Scholar
    3
    SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor
    Hoffmann, Markus; Kleine-Weber, Hannah; Schroeder, Simon; Krueger, Nadine; Herrler, Tanja; Erichsen, Sandra; Schiergens, Tobias S.; Herrler, Georg; Wu, Nai-Huei; Nitsche, Andreas; Mueller, Marcel A.; Drosten, Christian; Poehlmann, Stefan
    Cell (Cambridge, MA, United States) (2020), 181 (2), 271-280.e8CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clin. use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktl2qtb8%253D&md5=60aea5c939a2d4df034a91d6198fb3ef
  4. 4
    Wong, S. K.; Li, W.; Moore, M. J.; Choe, H.; Farzan, M. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J. Biol. Chem. 2004, 279, 3197– 3201,  DOI: 10.1074/jbc.C300520200
    Google Scholar
    4
    A 193-Amino Acid Fragment of the SARS Coronavirus S Protein Efficiently Binds Angiotensin-converting Enzyme 2
    Wong, Swee Kee; Li, Wenhui; Moore, Michael J.; Choe, Hyeryun; Farzan, Michael
    Journal of Biological Chemistry (2004), 279 (5), 3197-3201CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a crit. target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished assocn. of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC50 of less than 10 nM, whereas the IC50 of the S1 domain was ∼50 nM. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmtlCiug%253D%253D&md5=217bbd4071430c68b64095fc55ef247e
  5. 5
    Wang, D.; Baudys, J.; Bundy, J. L.; Solano, M.; Keppel, T.; Barr, J. R. Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry. Anal. Chem. 2020, 92, 14730– 14739,  DOI: 10.1021/acs.analchem.0c03301
    Google Scholar
    5
    Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry
    Wang, Dongxia; Baudys, Jakub; Bundy, Jonathan L.; Solano, Maria; Keppel, Theodore; Barr, John R.
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (21), 14730-14739CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric anal. of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissocn. (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were obsd. in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these 2 species. In addn., we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVyku7fE&md5=8522e95853566d451830875cc6cf7de8
  6. 6
    Sanda, M.; Morrison, L.; Goldman, R. N- and O-glycosylation of the SARS-CoV-2 spike protein. Anal Chem. 2021, 93, 2003– 2009,  DOI: 10.1021/acs.analchem.0c03173
    Google Scholar
    6
    N- and O-Glycosylation of the SARS-CoV-2 Spike Protein
    Sanda, Miloslav; Morrison, Lindsay; Goldman, Radoslav
    Analytical Chemistry (Washington, DC, United States) (2021), 93 (4), 2003-2009CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compds. and vaccines requires detailed descriptive studies of SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage site that differs from the SARS-CoV spike protein of 2002. By anal. of the protein produced in HEK293 cells, we observe that the spike is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addn., we have identified eight addnl. O-glycopeptides on the spike glycoprotein and confirmed that the spike protein is heavily N-glycosylated. Our recently developed liq. chromatog.-mass spectrometry methodol. allowed us to identify LacdiNAc structural motifs on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike protein's glycosylation and enables the investigation of the influence of O-glycosylation on its proteolytic activation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjslOktA%253D%253D&md5=1e13437daf40436003c791786791a3a3
  7. 7
    Uslupehlivan, M.; Şener, E. Glycoinformatics approach for identifying target positions to inhibit initial binding of SARS-CoV-2 S1 protein to the host cell. bioRxiv 2020,  DOI: 10.1101/2020.03.25.007898
    Google Scholar
    There is no corresponding record for this reference.
  8. 8
    Casalino, L.; Gaieb, Z.; Goldsmith, J. A.; Hjorth, C. K.; Dommer, A. C.; Harbison, A. M.; Fogarty, C. A.; Barros, E. P.; Taylor, B. C.; McLellan, J. S.; Fadda, E.; Amaro, R. E. Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein. ACS Cent. Sci. 2020, 6, 1722– 1734,  DOI: 10.1021/acscentsci.0c01056
    Google Scholar
    8
    Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein
    Casalino, Lorenzo; Gaieb, Zied; Goldsmith, Jory A.; Hjorth, Christy K.; Dommer, Abigail C.; Harbison, Aoife M.; Fogarty, Carl A.; Barros, Emilia P.; Taylor, Bryn C.; McLellan, Jason S.; Fadda, Elisa; Amaro, Rommie E.
    ACS Central Science (2020), 6 (10), 1722-1734CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biol. data. Multiple microsecond-long, all-atom mol. dynamics simulations were used to provide an atomistic perspective on the roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike's receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry expts., which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the "down" state. Addnl., end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this mol. machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development. The glycan shield is a sugary barrier that helps the viral SARS-CoV-2 spikes to evade the immune system. Beyond shielding, two of the spike's glycans are discovered to prime the virus for infection.
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  9. 9
    Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L.; Wang, X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 2020, 581, 215– 220,  DOI: 10.1038/s41586-020-2180-5
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    9
    Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
    Lan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, Xinquan
    Nature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
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  10. 10
    Mehdipour, A. R.; Hummer, G. Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike. bioRxiv 2020,  DOI: 10.1101/2020.07.09.193680
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    There is no corresponding record for this reference.
  11. 11
    Li, Q.; Wu, J.; Nie, J.; Zhang, L.; Hao, H.; Liu, S.; Zhao, C.; Zhang, Q.; Liu, H.; Nie, L.; Qin, H.; Wang, M.; Lu, Q.; Li, X.; Sun, Q.; Liu, J.; Zhang, L.; Li, X.; Huang, W.; Wang, Y. The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity. Cell 2020, 182, 1284– 1294.e9,  DOI: 10.1016/j.cell.2020.07.012
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    11
    The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity
    Li, Qianqian; Wu, Jiajing; Nie, Jianhui; Zhang, Li; Hao, Huan; Liu, Shuo; Zhao, Chenyan; Zhang, Qi; Liu, Huan; Nie, Lingling; Qin, Haiyang; Wang, Meng; Lu, Qiong; Li, Xiaoyu; Sun, Qiyu; Liu, Junkai; Zhang, Linqi; Li, Xuguang; Huang, Weijin; Wang, Youchun
    Cell (Cambridge, MA, United States) (2020), 182 (5), 1284-1294.e9CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biol. significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants contg. both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.
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  12. 12
    Premkumar, L.; Segovia-Chumbez, B.; Jadi, R.; Martinez, D. R.; Raut, R.; Markmann, A. J.; Cornaby, C.; Bartelt, L.; Weiss, S.; Park, Y.; Edwards, C. E.; Weimer, E.; Scherer, E. M.; Rouphael, N.; Edupuganti, S.; Weiskopf, D.; Tse, L. V.; Hou, Y. J.; Margolis, D.; Sette, A.; Collins, M. H.; Schmitz, J.; Baric, R. S.; de Silva, A. M. The receptor-binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci. Immunol. 2020, 5, eabc8413  DOI: 10.1126/sciimmunol.abc8413
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    There is no corresponding record for this reference.
  13. 13
    Pinto, D.; Park, Y.-J.; Beltramello, M.; Walls, A. C.; Tortorici, M. A.; Bianchi, S.; Jaconi, S.; Culap, K.; Zatta, F.; De Marco, A.; Peter, A.; Guarino, B.; Spreafico, R.; Cameroni, E.; Case, J. B.; Chen, R. E.; Havenar-Daughton, C.; Snell, G.; Telenti, A.; Virgin, H. W.; Lanzavecchia, A.; Diamond, M. S.; Fink, K.; Veesler, D.; Corti, D. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 2020, 583, 290– 295,  DOI: 10.1038/s41586-020-2349-y
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    13
    Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody
    Pinto, Dora; Park, Young-Jun; Beltramello, Martina; Walls, Alexandra C.; Tortorici, M. Alejandra; Bianchi, Siro; Jaconi, Stefano; Culap, Katja; Zatta, Fabrizia; De Marco, Anna; Peter, Alessia; Guarino, Barbara; Spreafico, Roberto; Cameroni, Elisabetta; Case, James Brett; Chen, Rita E.; Havenar-Daughton, Colin; Snell, Gyorgy; Telenti, Amalio; Virgin, Herbert W.; Lanzavecchia, Antonio; Diamond, Michael S.; Fink, Katja; Veesler, David; Corti, Davide
    Nature (London, United Kingdom) (2020), 583 (7815), 290-295CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in >3.7 million infections and 260,000 deaths as of 6 May 2020. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. We describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope contg. a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails contg. S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.
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  14. 14
    Zhou, D.; Tian, X.; Qi, R.; Peng, C.; Zhang, W. Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics. Glycobiology 2020, 31, 69– 80,  DOI: 10.1093/glycob/cwaa052
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    There is no corresponding record for this reference.
  15. 15
    Muhuri, M.; Gao, G. Is smaller better? Vaccine targeting recombinant receptor-binding domain might hold the key for mass production of effective prophylactics to fight the COVID-19 pandemic. Signal Transduction Targeted Ther. 2020, 5, 222  DOI: 10.1038/s41392-020-00317-1
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    15
    Is smaller better? Vaccine targeting recombinant receptor-binding domain might hold the key for mass production of effective prophylactics to fight the COVID-19 pandemic
    Muhuri, Manish; Gao, Guangping
    Signal Transduction and Targeted Therapy (2020), 5 (1), 222CODEN: STTTCB; ISSN:2059-3635. (Nature Research)
    A review. A recent report by Yang et al. published in Nature (https://doi.org/10.1038/s41586-020-2599-8 (2020)) reported a recombinant vaccine utilizing recombinant receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein. This vaccine candidate successfully induced potent functional antibody responses in the immunized mice, rabbits, and non-human primates. The study highlights the crit. role of the immunogenicity of the RBD domain upon SARS-CoV-2 infection and the alternate vaccine designs that could serve as effective prophylactics against the pandemic.
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  16. 16
    He, Y.; Zhou, Y.; Wu, H.; Luo, B.; Chen, J.; Li, W.; Jiang, S. Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines. J. Immunol. 2004, 173, 4050– 4057,  DOI: 10.4049/jimmunol.173.6.4050
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    16
    Identification of Immunodominant Sites on the Spike Protein of Severe Acute Respiratory Syndrome (SARS) Coronavirus: Implication for Developing SARS Diagnostics and Vaccines
    He, Yuxian; Zhou, Yusen; Wu, Hao; Luo, Baojun; Chen, Jingming; Li, Wanbo; Jiang, Shibo
    Journal of Immunology (2004), 173 (6), 4050-4057CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
    The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as detd. by Pepscan anal. with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S603-634, which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.
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  17. 17
    Lenza, M. P.; Oyenarte, I.; Diercks, T.; Quintana, J. I.; Gimeno, A.; Coelho, H.; Diniz, A.; Peccati, F.; Delgado, S.; Bosch, A.; Valle, M.; Millet, O.; Abrescia, N. G. A.; Palazón, A.; Marcelo, F.; Jiménez-Osés, G.; Jiménez-Barbero, J.; Ardá, A.; Ereño-Orbea, J. Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins. Angew. Chem., Int. Ed. 2020, 59, 23763– 23771,  DOI: 10.1002/anie.202011015
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    17
    Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins
    Lenza, Maria Pia; Oyenarte, Iker; Diercks, Tammo; Quintana, Jon Imanol; Gimeno, Ana; Coelho, Helena; Diniz, Ana; Peccati, Francesca; Delgado, Sandra; Bosch, Alexandre; Valle, Mikel; Millet, Oscar; Abrescia, Nicola G. A.; Palazon, Asis; Marcelo, Filipa; Jimenez-Oses, Gonzalo; Jimenez-Barbero, Jesus; Arda, Ana; Ereno-Orbea, June
    Angewandte Chemie, International Edition (2020), 59 (52), 23763-23771CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The glycan structures of the receptor-binding domain of the SARS-CoV-2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analyzed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD 13C-labeled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15N-labeled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary expts. from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.
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  18. 18
    Resemann, A.; Jabs, W.; Wiechmann, A.; Wagner, E.; Colas, O.; Evers, W.; Belau, E.; Vorwerg, L.; Evans, C.; Beck, A.; Suckau, D. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing. mAbs 2016, 8, 318– 330,  DOI: 10.1080/19420862.2015.1128607
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    18
    Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing
    Resemann, Anja; Jabs, Wolfgang; Wiechmann, Anja; Wagner, Elsa; Colas, Olivier; Evers, Waltraud; Belau, Eckhard; Vorwerg, Lars; Evans, Catherine; Beck, Alain; Suckau, Detlev
    mAbs (2016), 8 (2), 318-330CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
    The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the anal. data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called "bottom-up" approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall anal. time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their ref. sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced - the "Sequence Validation Percentage.". Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up mol. wt. detn. and middle-down protein sequencing. Three errors in the ref. amino acid sequence of natalizumab, causing a cumulative mass shift of only -2 Da in the natalizumab Fd domain, were cor. as a result of this work.
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  19. 19
    Gstöttner, C.; Reusch, D.; Haberger, M.; Dragan, I.; Van Veelen, P.; Kilgour, D. P. A.; Tsybin, Y. O.; van der Burgt, Y. E. M.; Wuhrer, M.; Nicolardi, S. Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry. mAbs 2020, 12, 1682403  DOI: 10.1080/19420862.2019.1682403
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    19
    Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry
    Gstottner Christoph; Dragan Irina; Van Veelen Peter; van der Burgt Yuri E M; Wuhrer Manfred; Nicolardi Simone; Reusch Dietmar; Haberger Markus; Kilgour David P A; Tsybin Yury O
    mAbs (2020), 12 (1), 1682403 ISSN:.
    Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.
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  20. 20
    Santos, M. R.; Ratnayake, C. K.; Fonslow, B.; Guttman, A. A Covalent, Cationic Polymer Coating Method for the CESI-MS Analysis of Intact Proteins and Polypeptides, SCIEX Separations, Brea, CA, 2015.
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    There is no corresponding record for this reference.
  21. 21
    Yang, Y.; Liu, F.; Franc, V.; Halim, L. A.; Schellekens, H.; Heck, A. J. R. Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity. Nat. Commun. 2016, 7, 13397  DOI: 10.1038/ncomms13397
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    21
    Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity
    Yang, Yang; Liu, Fan; Franc, Vojtech; Halim, Liem Andhyk; Schellekens, Huub; Heck, Albert J. R.
    Nature Communications (2016), 7 (), 13397CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resoln. native mass spectrometry and middle-down proteomics, to analyze this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addn. unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quant. assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.
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  22. 22
    Hinneburg, H.; Stavenhagen, K.; Schweiger-Hufnagel, U.; Pengelley, S.; Jabs, W.; Seeberger, P. H.; Silva, D. V.; Wuhrer, M.; Kolarich, D. The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics. J. Am. Soc. Mass Spectrom. 2016, 27, 507– 519,  DOI: 10.1007/s13361-015-1308-6
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    22
    The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics
    Hinneburg, Hannes; Stavenhagen, Kathrin; Schweiger-Hufnagel, Ulrike; Pengelley, Stuart; Jabs, Wolfgang; Seeberger, Peter H.; Silva, Daniel Varon; Wuhrer, Manfred; Kolarich, Daniel
    Journal of the American Society for Mass Spectrometry (2016), 27 (3), 507-519CODEN: JAMSEF; ISSN:1044-0305. (Springer)
    In-depth site-specific investigations of protein glycosylation are the basis for understanding the biol. function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable detn. of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied anal. tools, including glycopeptide fragmentation regimes and automated data anal. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissocn. (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS expt. We validated this method analyzing a set of human Igs (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compns., peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide anal. platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.
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  23. 23
    Zhang, T.; Madunić, K.; Holst, S.; Zhang, J.; Jin, C.; ten Dijke, P.; Karlsson, N. G.; Stavenhagen, K.; Wuhrer, M. Development of a 96-well plate sample preparation method for integrated N- and O-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry. Mol. Omics 2020, 16, 355– 363,  DOI: 10.1039/C9MO00180H
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    23
    Development of a 96-well plate sample preparation method for integrated N- and O-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry
    Zhang, Tao; Madunic, Katarina; Holst, Stephanie; Zhang, Jing; Jin, Chunsheng; ten Dijke, Peter; Karlsson, Niclas G.; Stavenhagen, Kathrin; Wuhrer, Manfred
    Molecular Omics (2020), 16 (4), 355-363CODEN: MOOMAW ISSN:. (Royal Society of Chemistry)
    Changes in glycosylation signatures of cells have been assocd. with pathol. processes in cancer as well as infectious and autoimmune diseases. The current protocols for comprehensive anal. of N-glycomics and O-glycomics derived from cells and tissues often require a large amt. of biol. material. They also only allow the processing of very limited nos. of samples at a time. Here we established a workflow for sequential release of N-glycans and O-glycans based on PVDF membrane immobilization in 96-well format from 5 x 105 cells. Released glycans are reduced, desalted, purified, and reconstituted, all in 96-well format plates, without addnl. staining or derivatization. The developed protocol allows the anal. of N- and O-glycans from relatively large nos. of samples in a less time consuming way with high repeatability. Inter- and intraday repeatability of the fetuin N-glycan anal. showed two median intraday coeffs. of variations (CVs) of 7.6% and 8.0%, and a median interday CV of 9.8%. Median CVs of 7.9% and 8.7% for the main peaks of N- and O-glycans released from the NMuMG cell line indicate a very good repeatability. The method is applicable to purified glycoproteins as well as to biofluids and cell- or tissue-based samples.
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  24. 24
    Madunić, K.; Zhang, T.; Mayboroda, O. A.; Holst, S.; Stavenhagen, K.; Jin, C.; Karlsson, N. G.; Lageveen-Kammeijer, G. S. M.; Wuhrer, M. Colorectal cancer cell lines show striking diversity of their O-glycome reflecting the cellular differentiation phenotype. Cell. Mol. Life Sci. 2021, 78, 337– 350,  DOI: 10.1007/s00018-020-03504-z
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    24
    Colorectal cancer cell lines show striking diversity of their O-glycome reflecting the cellular differentiation phenotype
    Madunic, Katarina; Zhang, Tao; Mayboroda, Oleg A.; Holst, Stephanie; Stavenhagen, Kathrin; Jin, Chunsheng; Karlsson, Niclas G.; Lageveen-Kammeijer, Guinevere S. M.; Wuhrer, Manfred
    Cellular and Molecular Life Sciences (2021), 78 (1), 337-350CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)
    Abstr.: Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their assocn. with other mol. features, an in-depth O-glycosylation anal. of 26 different CRC cell lines was performed. The released O-glycans were analyzed on porous graphitized carbon nano-liq. chromatog. system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC-ESI-MS/MS) allowing isomeric sepn. as well as in-depth structural characterization. Assocns. between the obsd. glycan phenotypes with previously reported cell line transcriptome signatures were examd. by canonical correlation anal. Striking differences are obsd. between the O-glycomes of 26 CRC cell lines. Unsupervized principal component anal. reveals a sepn. between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated α2,6-core sialylated glycans. Moreover, the expression of glycan signatures assocs. with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies.
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  25. 25
    Karlsson, N. G.; Wilson, N. L.; Wirth, H. J.; Dawes, P.; Joshi, H.; Packer, N. H. Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis. Rapid Commun. Mass Spectrom. 2004, 18, 2282– 2292,  DOI: 10.1002/rcm.1626
    Google Scholar
    25
    Negative ion graphitized carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis
    Karlsson, Niclas G.; Wilson, Nicole L.; Wirth, Hans-Juergen; Dawes, Peter; Joshi, Hiren; Packer, Nicolle H.
    Rapid Communications in Mass Spectrometry (2004), 18 (19), 2282-2292CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Neg. ion nano-liq. chromatog./mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS2), using graphitized carbon as sepg. medium, were explored for analyzing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 μL/min) coupled with a conventional electrospray ionization source, the nano-LC/MS (flow rate of 0.6 μL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The abs. signals for the [M-2H]n- ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS2 mode, in which detection of low abundant sequence ions is necessary for characterization of highly sialylated N-linked oligosaccharides. Oligosaccharides with high nos. of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS2 anal. was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitized carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 μg of total amt. of protein).
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  26. 26
    Karlsson, N. G.; Schulz, B. L.; Packer, N. H. Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J. Am. Soc. Mass Spectrom. 2004, 15, 659– 672,  DOI: 10.1016/j.jasms.2004.01.002
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    26
    Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn
    Karlsson, Niclas G.; Schulz, Benjamin L.; Packer, Nicolle H.
    Journal of the American Society for Mass Spectrometry (2004), 15 (5), 659-672CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
    Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were sepd. and detected by neg. ion LC-MS and LC-MS2. The resoln. of the chromatog. and the information obtained from collision induced dissocn. of detected [M - H]- ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nona-saccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Zi and Yi), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAcβ1-3GalNAcol or Gal/GlcNAcβ1-3(GlcNAcβ1-6)GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and Ai type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution (0,2Ai - H2O cleavage) and disubstitution of C-3 and C-4 (Zi/Zi cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Galβ1-3/4GlcNAcβ1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, neg. ion LC-MS2 was able to generate extensive data for understanding the overall glycosylation pattern of a sample, esp. when only a limited amt. of material is available.
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  27. 27
    Anugraham, M.; Everest-Dass, A. V.; Jacob, F.; Packer, N. H. A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells. Rapid Commun. Mass Spectrom. 2015, 29, 545– 561,  DOI: 10.1002/rcm.7130
    Google Scholar
    27
    A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells
    Anugraham, Merrina; Everest-Dass, Arun Vijay; Jacob, Francis; Packer, Nicolle H.
    Rapid Communications in Mass Spectrometry (2015), 29 (7), 545-561CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Glycosphingolipids (GSLs) constitute a highly diverse class of glyco-conjugates which are involved in many aspects of cell membrane function and disease. The isolation, detection and structural characterization of the carbohydrate (glycan) component of GSLs are particularly challenging given their structural heterogeneity and thus rely on the development of sensitive, anal. technologies. Neutral and acidic GSL stds. were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymically released using endoglycoceramidase II (EGCase II), sepd. by porous graphitized carbon (PGC) liq. chromatog. and structurally characterized by neg. ion mode electrospray ionization tandem mass spectrometry (PGC-LC/ESI-MS/MS). This approach was then employed for GSLs isolated from 100 mg of serous and endometrioid cancer tissue and from cell line (107 cells) samples. Glycans were released from GSL stds. comprised of ganglio-, asialo-ganglio- and the relatively resistant globo-series glycans, using as little as 1 mU of enzyme and 2 μg of GSL. The platform of anal. was then applied to GSLs isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatog. resolved on PGC and characterized by comparison with the MS2 fragment ion spectra of the glycan stds. and by application of known structural MS2 fragment ions. This approach identified several (neo-)lacto-, globo- and ganglio-series glycans and facilitated the discrimination of isomeric structures contg. Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides. Thus, the authors describe a relatively simple, detergent-free, enzymic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural anal. afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the anal. of tumor and cell-derived GSL-glycans. The method uses the potential of MS2 fragmentation in neg. ion ESI mode to characterize, in detail, the biol. relevant glycan structures derived from GSLs.
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  28. 28
    Ceroni, A.; Maass, K.; Geyer, H.; Geyer, R.; Dell, A.; Haslam, S. M. GlycoWorkbench: A Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans. J. Proteome Res. 2008, 7, 1650– 1659,  DOI: 10.1021/pr7008252
    Google Scholar
    28
    GlycoWorkbench: A Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans
    Ceroni, Alessio; Maass, Kai; Geyer, Hildegard; Geyer, Rudolf; Dell, Anne; Haslam, Stuart M.
    Journal of Proteome Research (2008), 7 (4), 1650-1659CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Mass spectrometry is the main anal. technique currently used to address the challenges of glycomics as it offers unrivaled levels of sensitivity and the ability to handle complex mixts. of different glycan variations. Detn. of glycan structures from anal. of MS data is a major bottleneck in high-throughput glycomics projects, and robust solns. to this problem are of crit. importance. However, all the approaches currently available have inherent restrictions to the type of glycans they can identify, and none of them have proved to be a definitive tool for glycomics. GlycoWorkbench is a software tool developed by the EUROCarbDB initiative to assist the manual interpretation of MS data. The main task of GlycoWorkbench is to evaluate a set of structures proposed by the user by matching the corresponding theor. list of fragment masses against the list of peaks derived from the spectrum. The tool provides an easy to use graphical interface, a comprehensive and increasing set of structural constituents, an exhaustive collection of fragmentation types, and a broad list of annotation options. The aim of GlycoWorkbench is to offer complete support for the routine interpretation of MS data. The software is available for download from: http://www.eurocarbdb.org/applications/ms-tools.
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  29. 29
    Cooper, C. A.; Gasteiger, E.; Packer, N. H. GlycoMod--a software tool for determining glycosylation compositions from mass spectrometric data. Proteomics 2001, 1, 340– 349,  DOI: 10.1002/1615-9861(200102)1:2<340::AID-PROT340>3.0.CO;2-B
    Google Scholar
    29
    GlycoMod - A software tool for determining glycosylation compositions from mass spectrometric data
    Cooper, Catherine A.; Gasteiger, Elisabeth; Packer, Nicolle H.
    Proteomics (2001), 1 (2), 340-349CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH)
    GlycoMod is a software tool designed to find all possible compns. of a glycan structure from its exptl. detd. mass. The program can be used to predict the compn. of any glycoprotein-derived oligosaccharide comprised of either underivatized, methylated or acetylated monosaccharides, or with a derivatized reducing terminus. The compn. of a glycan attached to a peptide can be computed if the sequence or mass of the peptide is known. In addn., if the protein is known and is contained in the SWISS-PROT or TrEMBL databases, the program will match the exptl. detd. masses against all the predicted protease-produced peptides (including any post-translational modifications annotated in these databases) which have the potential to be glycosylated with either N- or O-linked oligosaccharides. Since many possible glycan compns. can be generated from the same mass, the program can apply compositional constraints to the output if the user supplies either known or suspected monosaccharide constituents. Furthermore, known oligosaccharide structural constraints on monosaccharide compn. are also incorporated into the program to limit the output.
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  30. 30
    Wohlschlager, T.; Scheffler, K.; Forstenlehner, I. C.; Skala, W.; Senn, S.; Damoc, E.; Holzmann, J.; Huber, C. G. Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals. Nat. Commun. 2018, 9, 1713  DOI: 10.1038/s41467-018-04061-7
    Google Scholar
    30
    Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals
    Wohlschlager Therese; Forstenlehner Ines C; Skala Wolfgang; Senn Stefan; Huber Christian G; Wohlschlager Therese; Scheffler Kai; Forstenlehner Ines C; Skala Wolfgang; Senn Stefan; Holzmann Johann; Huber Christian G; Scheffler Kai; Forstenlehner Ines C; Holzmann Johann; Damoc Eugen
    Nature communications (2018), 9 (1), 1713 ISSN:.
    Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom-up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.
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  31. 31
    Hati, S.; Bhattacharyya, S. Impact of Thiol-Disulfide Balance on the Binding of Covid-19 Spike Protein with Angiotensin-Converting Enzyme 2 Receptor. ACS Omega 2020, 5, 16292– 16298,  DOI: 10.1021/acsomega.0c02125
    Google Scholar
    31
    Impact of Thiol-Disulfide Balance on the Binding of Covid-19 Spike Protein with Angiotensin-Converting Enzyme 2 Receptor
    Hati, Sanchita; Bhattacharyya, Sudeep
    ACS Omega (2020), 5 (26), 16292-16298CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an ongoing pandemic of coronavirus disease (COVID-19), which started in 2019. This is a member of Coronaviridae family in the genus Betacoronavirus, which also includes SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). The angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV and SARS-CoV-2 to enter the host cells. In particular, the interaction of viral spike proteins with ACE2 is a crit. step in the viral replication cycle. The receptor-binding domain of the viral spike proteins and ACE2 have several cysteine residues. In this study, the role of thiol-disulfide balance on the interactions between SARS-CoV/CoV-2 spike proteins and ACE2 was investigated using mol. dynamics simulations. The study revealed that the binding affinity was significantly impaired when all of the disulfide bonds of both ACE2 and SARS-CoV/CoV-2 spike proteins were reduced to thiol groups. The impact on the binding affinity was less severe when the disulfide bridges of only one of the binding partners were reduced to thiols. This computational finding possibly provides a mol. basis for the differential COVID-19 cellular recognition due to the oxidative stress.
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  32. 32
    Gstöttner, C.; Nicolardi, S.; Haberger, M.; Reusch, D.; Wuhrer, M.; Domínguez-Vega, E. Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS. Anal. Chim. Acta 2020, 1134, 18– 27,  DOI: 10.1016/j.aca.2020.07.069
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    32
    Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS
    Gstottner, Christoph; Nicolardi, Simone; Haberger, Markus; Reusch, Dietmar; Wuhrer, Manfred; Dominguez-Vega, Elena
    Analytica Chimica Acta (2020), 1134 (), 18-27CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be obsd. contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new anal. strategies are needed for a more accurate and in-depth anal. Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a pos. coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the sepn. permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond redn. The six different subunits (Lc1, Lc2, Fd'1, Fd'2, (Fc/2)1 and (Fc/2)2) were sepd. using the same pos.-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resoln. (resolving power 140000 at m/z 1100) for a more confident anal. of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately detd. in the subunits showing the complementarity of both approaches.
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  33. 33
    Trastoy, B.; Naegeli, A.; Anso, I.; Sjögren, J.; Guerin, M. E. Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila. Nat. Commun. 2020, 11, 4844  DOI: 10.1038/s41467-020-18696-y
    Google Scholar
    33
    Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila
    Trastoy, Beatriz; Naegeli, Andreas; Anso, Itxaso; Sjogren, Jonathan; Guerin, Marcelo E.
    Nature Communications (2020), 11 (1), 4844CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the human gut that promotes a beneficial effect on health, likely based on the regulation of mucus thickness and gut barrier integrity, but also on the modulation of the immune system. In this work, we focus in OgpA from A. muciniphila, an O-glycopeptidase that exclusively hydrolyzes the peptide bond N-terminal to serine or threonine residues substituted with an O-glycan. We det. the high-resoln. X-ray crystal structures of the unliganded form of OgpA, the complex with the glycodrosocin O-glycopeptide substrate and its product, providing a comprehensive set of snapshots of the enzyme along the catalytic cycle. In combination with O-glycopeptide chem., enzyme kinetics, and computational methods we unveil the mol. mechanism of O-glycan recognition and specificity for OgpA. The data also contribute to understanding how A. muciniphila processes mucins in the gut, as well as anal. of post-translational O-glycosylation events in proteins.
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  34. 34
    Guile, G. R.; Harvey, D. J.; O’Donnell, N.; Powell, A. K.; Hunter, A. P.; Zamze, S.; Fernandes, D. L.; Dwek, R. A.; Wing, D. R. Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland. Eur. J. Biochem. 1998, 258, 623– 656,  DOI: 10.1046/j.1432-1327.1998.2580623.x
    Google Scholar
    34
    Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland
    Guile, Geoffrey R.; Harvey, David J.; O'Donnell, Niall; Powell, Andrew K.; Hunter, Ann P.; Zamze, Susanne; Fernandes, Daryl L.; Dwek, Raymond A.; Wing, David R.
    European Journal of Biochemistry (1998), 258 (2), 623-656CODEN: EJBCAI; ISSN:0014-2956. (Springer-Verlag)
    The glycosylation of a no. of constituents of human saliva is known to modify its biol. roles, such as its lubricating properties and binding of microbial flora. Gillece-Castro et al. [Gillece-Castro, B. L., Prakobphol, A., Burlingame, A. L., Leffler, H. & Fisher, S. J. (1991) J. Biol. Chem. 266, 17358-17368] have proposed that the major glycan on the salivary proline-rich glycoproteins is a trifucosylated biantennary sugar with one difucosylated and one unfucosylated antenna. Furthermore, they proposed that the non-fucosylated antenna mediated adherence to a periodontal pathogen, Fusobacterium nucleatum. The detailed structures and roles of other highly fucosylated glycans that co-exist in the parotid gland are not fully known. In view of the influence of outer-arm fucosylation on carbohydrate recognition processes in general, this paper reports the use of a combination of HPLC (normal and reversed phase), matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry and exoglycosidase digestions to dissect the detailed structures of the most abundant of these polyfucosylated glycans. For measurement of reversed-phase HPLC retention times, new calibration units were used which paralleled the glucose units used for normal-phase HPLC. These differed in that the difference in retention times were compared with those derived from a ladder of 2-aminobenzamide-labeled arabinose oligomers instead of the corresponding oligomers from partially hydrolyzed dextran. Over sixty neutral sugars were identified from the parotid gland and many of these were addnl. found substituted with sialic acid (both α2-3-linked and α2-6-linked) and sulfate. These glycans were mainly bi- and tri-antennary sugars with up to five and seven fucose residues resp., contg. fucose α1-3-linked to the outer-arm GlcNAc residues and α1-2-linked to the galactose. All fucosylated structures contained a core (α1-6-linked) fucose. The detailed structure of the trifucosylated biantennary glycan was confirmed, together with the structures of another 12 fucosylated biantennary glycans. Smaller amts. of hybrid and tetraantennary structures were also found and bisected glycans were shown to be constituents of parotid glycoproteins for the first time. Acidic glycans were mainly substituted with sialic acid. Most were monosialylated as the presence of fucose on the antennae was found to suppress the addn. of extra sialic acid moieties. The possible functional significance of highly fucosylated N-glycans is discussed in relation to their modification of the availability of other non-reducing terminal monosaccharides for recognition processes.
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  2. Bojing Zhu, Zexuan Chen, Jiechen Shen, Yintai Xu, Rongxia Lan, Shisheng Sun. Structural- and Site-Specific N-Glycosylation Characterization of COVID-19 Virus Spike with StrucGP. Analytical Chemistry 2022, 94 (36) , 12274-12279. https://doi.org/10.1021/acs.analchem.2c02265
  3. Ebba Samuelsson, Ekaterina Mirgorodskaya, Kristina Nyström, Malin Bäckström, Jan-Åke Liljeqvist, Rickard Nordén. Sialic Acid and Fucose Residues on the SARS-CoV-2 Receptor-Binding Domain Modulate IgG Antibody Reactivity. ACS Infectious Diseases 2022, 8 (9) , 1883-1893. https://doi.org/10.1021/acsinfecdis.2c00155
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  6. Joel D. Allen, Himanshi Chawla, Firdaus Samsudin, Lorena Zuzic, Aishwary Tukaram Shivgan, Yasunori Watanabe, Wan-ting He, Sean Callaghan, Ge Song, Peter Yong, Philip J. M. Brouwer, Yutong Song, Yongfei Cai, Helen M. E. Duyvesteyn, Tomas Malinauskas, Joeri Kint, Paco Pino, Maria J. Wurm, Martin Frank, Bing Chen, David I. Stuart, Rogier W. Sanders, Raiees Andrabi, Dennis R. Burton, Sai Li, Peter J. Bond, Max Crispin. Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation. Biochemistry 2021, 60 (27) , 2153-2169. https://doi.org/10.1021/acs.biochem.1c00279
  7. Itxaso Anso, Andreas Naegeli, Javier O. Cifuente, Ane Orrantia, Erica Andersson, Olatz Zenarruzabeitia, Alicia Moraleda-Montoya, Mikel García-Alija, Francisco Corzana, Rafael A. Del Orbe, Francisco Borrego, Beatriz Trastoy, Jonathan Sjögren, Marcelo E. Guerin. Turning universal O into rare Bombay type blood. Nature Communications 2023, 14 (1) https://doi.org/10.1038/s41467-023-37324-z
  8. Janelle Sauvageau, Izel Koyuturk, Frank St. Michael, Denis Brochu, Marie-France Goneau, Ian Schoenhofen, Sylvie Perret, Alexandra Star, Anna Robotham, Arsalan Haqqani, John Kelly, Michel Gilbert, Yves Durocher. Simplifying glycan monitoring of complex antigens such as the SARS-CoV-2 spike to accelerate vaccine development. Communications Chemistry 2023, 6 (1) https://doi.org/10.1038/s42004-023-00988-1
  9. Federico Carrión, Florencia Rammauro, Natalia Olivero‐Deibe, Martín Fló, María Magdalena Portela, Analía Lima, Rosario Durán, Otto Pritsch, Sergio Bianchi. Soluble SARS‐CoV ‐2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface. Protein Science 2023, 32 (8) https://doi.org/10.1002/pro.4721
  10. Deseree J. Reid, Stephanie Thibert, Mowei Zhou. Dissecting the structural heterogeneity of proteins by native mass spectrometry. Protein Science 2023, 32 (4) https://doi.org/10.1002/pro.4612
  11. Haiying Que, Li Chen, Xiawei Wei. SARS-CoV-2 variants, immune escape, COVID-19 vaccine, and therapeutic strategies. Science China Life Sciences 2023, 66 (2) , 406-410. https://doi.org/10.1007/s11427-021-2164-6
  12. Huifang Xu, Tiantian Wang, Peng Sun, Xuchen Hou, Xin Gong, Bin Zhang, Jun Wu, Bo Liu. A bivalent subunit vaccine efficiently produced in Pichia pastoris against SARS-CoV-2 and emerging variants. Frontiers in Microbiology 2023, 13 https://doi.org/10.3389/fmicb.2022.1093080
  13. Arasu Balasubramaniyam, Emma Ryan, Dallas Brown, Therwa Hamza, William Harrison, Michael Gan, Rajeshwer S. Sankhala, Wei-Hung Chen, Elizabeth J. Martinez, Jaime L. Jensen, Vincent Dussupt, Letzibeth Mendez-Rivera, Sandra Mayer, Jocelyn King, Nelson L. Michael, Jason Regules, Shelly Krebs, Mangala Rao, Gary R. Matyas, M. Gordon Joyce, Adrian H. Batchelor, Gregory D. Gromowski, Sheetij Dutta. Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain (RBD) Produced in E. coli Combined with the Army Liposomal Formulation Containing QS21 (ALFQ) Elicits Neutralizing Antibodies against Mismatched Variants. Vaccines 2023, 11 (1) , 42. https://doi.org/10.3390/vaccines11010042
  14. María Celeste Rodríguez, Natalia Ceaglio, Agustina Gugliotta, Javier Villarraza, Ernesto Garay, Antonela Fuselli, Victoria Gastaldi, María Belén Tardivo, Sebastián Antuña, Diego Fontana, Claudio Prieto. Design and optimization of an IgG human ELISA assay reactive to recombinant RBD SARS-CoV-2 protein. Applied Microbiology and Biotechnology 2022, 106 (23) , 7933-7948. https://doi.org/10.1007/s00253-022-12254-w
  15. Sayantani Chatterjee, Joseph Zaia. Proteomics‐based mass spectrometry profiling of SARS‐CoV‐2 infection from human nasopharyngeal samples. Mass Spectrometry Reviews 2022, 25 https://doi.org/10.1002/mas.21813
  16. Termsak Tantiwiwat, Apisitt Thaiprayoon, Ake-kavitch Siriatcharanon, Chakrit Tachaapaikoon, Nongluk Plongthongkum, Dujduan Waraho-Zhmayev. Utilization of Receptor-Binding Domain of SARS-CoV-2 Spike Protein Expressed in Escherichia coli for the Development of Neutralizing Antibody Assay. Molecular Biotechnology 2022, 17 https://doi.org/10.1007/s12033-022-00563-4
  17. Diana Campos, Michael Girgis, Miloslav Sanda. Site‐specific glycosylation of SARS‐CoV‐2: Big challenges in mass spectrometry analysis. PROTEOMICS 2022, 22 (15-16) https://doi.org/10.1002/pmic.202100322
  18. Bo Liu, Ying Yin, Yuxiao Liu, Tiantian Wang, Peng Sun, Yangqin Ou, Xin Gong, Xuchen Hou, Jun Zhang, Hongguang Ren, Shiqiang Luo, Qian Ke, Yongming Yao, Junjie Xu, Jun Wu. A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses. Engineering 2022, 13 , 107-115. https://doi.org/10.1016/j.eng.2021.06.012
  19. Di Wu, Carol V. Robinson. Understanding glycoprotein structural heterogeneity and interactions: Insights from native mass spectrometry. Current Opinion in Structural Biology 2022, 74 , 102351. https://doi.org/10.1016/j.sbi.2022.102351
  20. Ying Sheng, Anita Vinjamuri, Michael Russelle S. Alvarez, Yixuan Xie, Marisa McGrath, Siyu Chen, Mariana Barboza, Matthew Frieman, Carlito B. Lebrilla. Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites. Frontiers in Molecular Biosciences 2022, 9 https://doi.org/10.3389/fmolb.2022.799703
  21. Chu-Wei Kuo, Tzu-Jing Yang, Yu-Chun Chien, Pei-Yu Yu, Shang-Te Danny Hsu, Kay-Hooi Khoo. Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants. Glycobiology 2022, 32 (1) , 60-72. https://doi.org/10.1093/glycob/cwab102
  22. Luis Ariel Espinosa, Yassel Ramos, Ivan Andújar, Enso Onill Torres, Gleysin Cabrera, Alejandro Martín, Diamilé Roche, Glay Chinea, Mónica Becquet, Isabel González, Camila Canaán-Haden, Elías Nelson, Gertrudis Rojas, Beatriz Pérez-Massón, Dayana Pérez-Martínez, Tamy Boggiano, Julio Palacio, Sum Lai Lozada Chang, Lourdes Hernández, Kathya Rashida de la Luz Hernández, Saloheimo Markku, Marika Vitikainen, Yury Valdés-Balbín, Darielys Santana-Medero, Daniel G. Rivera, Vicente Vérez-Bencomo, Mark Emalfarb, Ronen Tchelet, Gerardo Guillén, Miladys Limonta, Eulogio Pimentel, Marta Ayala, Vladimir Besada, Luis Javier González. In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI–MS spectrum. Analytical and Bioanalytical Chemistry 2021, 413 (30) , 7559-7585. https://doi.org/10.1007/s00216-021-03721-w
  23. Yanqiu Gong, Suideng Qin, Lunzhi Dai, Zhixin Tian. The glycosylation in SARS-CoV-2 and its receptor ACE2. Signal Transduction and Targeted Therapy 2021, 6 (1) https://doi.org/10.1038/s41392-021-00809-8
  24. Jinkai Zang, Yuanfei Zhu, Yu Zhou, Chenjian Gu, Yufang Yi, Shuxia Wang, Shiqi Xu, Gaowei Hu, Shujuan Du, Yannan Yin, Yalei Wang, Yong Yang, Xueyang Zhang, Haikun Wang, Feifei Yin, Chao Zhang, Qiang Deng, Youhua Xie, Zhong Huang. Yeast-produced RBD-based recombinant protein vaccines elicit broadly neutralizing antibodies and durable protective immunity against SARS-CoV-2 infection. Cell Discovery 2021, 7 (1) https://doi.org/10.1038/s41421-021-00315-9
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  • Abstract

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    Figure 1

    Figure 1. Multilevel characterization of CHO- and HEK293-RBD. Next to intact protein analysis using CE-MS and MALDI-ISD-MS, the samples are structurally characterized by glycan dissection, glycopeptide analysis, and released glycan analysis. Their functional characterization was performed by measuring their binding characteristics to ACE2 and anti-SARS-CoV-2 antibodies.

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    Figure 2

    Figure 2. Deconvoluted ESI mass spectra of the main peak observed after sheathless CE-MS for (A) CHO-RBD and (B) HEK293-RBD. HEK293-RBD carries an additional N-terminal pyroglutamate. Blue square, N-acetylglucosamine; yellow square, N-acetylgalactosamine; and yellow circle, galactose.

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    Figure 3

    Figure 3. PGC nano-LC-ESI-MS/MS of released O-glycans from (A) CHO-RBD and (B) HEK293-RBD. Yellow square, N-acetylgalactosamine; yellow circle, galactose; blue square, N-acetylglucosamine; red triangle, fucose; purple diamond, N-acetylneuraminic acid (sialic acid); and S, sulfate.

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    Figure 4

    Figure 4. Deconvoluted mass spectra of the intact RBD (not enzymatically treated) produced by HEK293 cells. The assignments were based on previous enzyme treatments, mass, and glycopeptide as well as released O-glycan data. Peaks marked with an asterisk * are presumably the acetylated variant of RBD. Yellow square, N-acetylgalactosamine; yellow circle, galactose; blue square, N-acetylglucosamine; red triangle, fucose; and purple diamond, N-acetylneuraminic acid (sialic acid).

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    Figure 5

    Figure 5. Deconvoluted mass spectra of the RBD intact (nonenzymatical treated) (black trace) and in silico simulated mass spectrum (vermillion trace) of RBD produced in (A) CHO cells or (B) HEK293 cells.

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    Figure 6

    Figure 6. ACE2 binding assay. Dose–response curves of CHO- or HEK293-RBD binding to the ACE2 receptor. Comparison to (A) deglycosylated version of CHO- or HEK293-RBD and (B) S or S1 subunit (produced in HEK293 cells).

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  • References

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    This article references 34 other publications.

    1. 1
      Li, H.; Liu, S.-M.; Yu, X.-H.; Tang, S.-L.; Tang, C.-K. Coronavirus disease 2019 (COVID-19): current status and future perspectives. Int. J. Antimicrob. Agents 2020, 55, 105951  DOI: 10.1016/j.ijantimicag.2020.105951
      1
      Coronavirus disease 2019 (COVID-19): current status and future perspectives
      Li, Heng; Liu, Shang-Ming; Yu, Xiao-Hua; Tang, Shi-Lin; Tang, Chao-Ke
      International Journal of Antimicrobial Agents (2020), 55 (5), 105951CODEN: IAAGEA; ISSN:0924-8579. (Elsevier B.V.)
      A review. Coronavirus disease 2019 (COVID-19) originated in the city of Wuhan, Hubei Province, Central China, and has spread quickly to 72 countries to date. COVID-19 is caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [previously provisionally known as 2019 novel coronavirus (2019-nCoV)]. At present, the newly identified SARS-CoV-2 has caused a large no. of deaths with tens of thousands of confirmed cases worldwide, posing a serious threat to public health. However, there are no clin. approved vaccines or specific therapeutic drugs available for COVID-19. Intensive research on the newly emerged SARS-CoV-2 is urgently needed to elucidate the pathogenic mechanisms and epidemiol. characteristics and to identify potential drug targets, which will contribute to the development of effective prevention and treatment strategies. Hence, this review will focus on recent progress regarding the structure of SARS-CoV-2 and the characteristics of COVID-19, such as the etiol., pathogenesis and epidemiol. characteristics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmvFSksLs%253D&md5=c4a9e77b6383b16414b6969186015854
    2. 2
      Watanabe, Y.; Allen, J. D.; Wrapp, D.; McLellan, J. S.; Crispin, M. Site-specific glycan analysis of the SARS-CoV-2 spike. Science 2020, 369, 330– 333,  DOI: 10.1126/science.abb9983
      2
      Site-specific glycan analysis of the SARS-CoV-2 spike
      Watanabe, Yasunori; Allen, Joel D.; Wrapp, Daniel; McLellan, Jason S.; Crispin, Max
      Science (Washington, DC, United States) (2020), 369 (6501), 330-333CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This anal. enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiol. and vaccine design.
      >> More from SciFinder ®
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    3. 3
      Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S.; Schiergens, T. S.; Herrler, G.; Wu, N.-H.; Nitsche, A.; Müller, M. A.; Drosten, C.; Pöhlmann, S. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020, 181, 271– 280.e8,  DOI: 10.1016/j.cell.2020.02.052
      3
      SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor
      Hoffmann, Markus; Kleine-Weber, Hannah; Schroeder, Simon; Krueger, Nadine; Herrler, Tanja; Erichsen, Sandra; Schiergens, Tobias S.; Herrler, Georg; Wu, Nai-Huei; Nitsche, Andreas; Mueller, Marcel A.; Drosten, Christian; Poehlmann, Stefan
      Cell (Cambridge, MA, United States) (2020), 181 (2), 271-280.e8CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clin. use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktl2qtb8%253D&md5=60aea5c939a2d4df034a91d6198fb3ef
    4. 4
      Wong, S. K.; Li, W.; Moore, M. J.; Choe, H.; Farzan, M. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J. Biol. Chem. 2004, 279, 3197– 3201,  DOI: 10.1074/jbc.C300520200
      4
      A 193-Amino Acid Fragment of the SARS Coronavirus S Protein Efficiently Binds Angiotensin-converting Enzyme 2
      Wong, Swee Kee; Li, Wenhui; Moore, Michael J.; Choe, Hyeryun; Farzan, Michael
      Journal of Biological Chemistry (2004), 279 (5), 3197-3201CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a crit. target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished assocn. of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC50 of less than 10 nM, whereas the IC50 of the S1 domain was ∼50 nM. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.
      >> More from SciFinder ®
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    5. 5
      Wang, D.; Baudys, J.; Bundy, J. L.; Solano, M.; Keppel, T.; Barr, J. R. Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry. Anal. Chem. 2020, 92, 14730– 14739,  DOI: 10.1021/acs.analchem.0c03301
      5
      Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry
      Wang, Dongxia; Baudys, Jakub; Bundy, Jonathan L.; Solano, Maria; Keppel, Theodore; Barr, John R.
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (21), 14730-14739CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric anal. of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissocn. (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were obsd. in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these 2 species. In addn., we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.
      >> More from SciFinder ®
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    6. 6
      Sanda, M.; Morrison, L.; Goldman, R. N- and O-glycosylation of the SARS-CoV-2 spike protein. Anal Chem. 2021, 93, 2003– 2009,  DOI: 10.1021/acs.analchem.0c03173
      6
      N- and O-Glycosylation of the SARS-CoV-2 Spike Protein
      Sanda, Miloslav; Morrison, Lindsay; Goldman, Radoslav
      Analytical Chemistry (Washington, DC, United States) (2021), 93 (4), 2003-2009CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compds. and vaccines requires detailed descriptive studies of SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage site that differs from the SARS-CoV spike protein of 2002. By anal. of the protein produced in HEK293 cells, we observe that the spike is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addn., we have identified eight addnl. O-glycopeptides on the spike glycoprotein and confirmed that the spike protein is heavily N-glycosylated. Our recently developed liq. chromatog.-mass spectrometry methodol. allowed us to identify LacdiNAc structural motifs on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike protein's glycosylation and enables the investigation of the influence of O-glycosylation on its proteolytic activation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXjslOktA%253D%253D&md5=1e13437daf40436003c791786791a3a3
    7. 7
      Uslupehlivan, M.; Şener, E. Glycoinformatics approach for identifying target positions to inhibit initial binding of SARS-CoV-2 S1 protein to the host cell. bioRxiv 2020,  DOI: 10.1101/2020.03.25.007898
      There is no corresponding record for this reference.
    8. 8
      Casalino, L.; Gaieb, Z.; Goldsmith, J. A.; Hjorth, C. K.; Dommer, A. C.; Harbison, A. M.; Fogarty, C. A.; Barros, E. P.; Taylor, B. C.; McLellan, J. S.; Fadda, E.; Amaro, R. E. Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein. ACS Cent. Sci. 2020, 6, 1722– 1734,  DOI: 10.1021/acscentsci.0c01056
      8
      Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein
      Casalino, Lorenzo; Gaieb, Zied; Goldsmith, Jory A.; Hjorth, Christy K.; Dommer, Abigail C.; Harbison, Aoife M.; Fogarty, Carl A.; Barros, Emilia P.; Taylor, Bryn C.; McLellan, Jason S.; Fadda, Elisa; Amaro, Rommie E.
      ACS Central Science (2020), 6 (10), 1722-1734CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biol. data. Multiple microsecond-long, all-atom mol. dynamics simulations were used to provide an atomistic perspective on the roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike's receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry expts., which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the "down" state. Addnl., end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this mol. machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development. The glycan shield is a sugary barrier that helps the viral SARS-CoV-2 spikes to evade the immune system. Beyond shielding, two of the spike's glycans are discovered to prime the virus for infection.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvVOlsb3N&md5=52d499afcd7e3caa7d9e6017ffa86e45
    9. 9
      Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L.; Wang, X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 2020, 581, 215– 220,  DOI: 10.1038/s41586-020-2180-5
      9
      Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
      Lan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, Xinquan
      Nature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtL8%253D&md5=279c60143e8e5eb505457e0778baa8ef
    10. 10
      Mehdipour, A. R.; Hummer, G. Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike. bioRxiv 2020,  DOI: 10.1101/2020.07.09.193680
      There is no corresponding record for this reference.
    11. 11
      Li, Q.; Wu, J.; Nie, J.; Zhang, L.; Hao, H.; Liu, S.; Zhao, C.; Zhang, Q.; Liu, H.; Nie, L.; Qin, H.; Wang, M.; Lu, Q.; Li, X.; Sun, Q.; Liu, J.; Zhang, L.; Li, X.; Huang, W.; Wang, Y. The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity. Cell 2020, 182, 1284– 1294.e9,  DOI: 10.1016/j.cell.2020.07.012
      11
      The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity
      Li, Qianqian; Wu, Jiajing; Nie, Jianhui; Zhang, Li; Hao, Huan; Liu, Shuo; Zhao, Chenyan; Zhang, Qi; Liu, Huan; Nie, Lingling; Qin, Haiyang; Wang, Meng; Lu, Qiong; Li, Xiaoyu; Sun, Qiyu; Liu, Junkai; Zhang, Linqi; Li, Xuguang; Huang, Weijin; Wang, Youchun
      Cell (Cambridge, MA, United States) (2020), 182 (5), 1284-1294.e9CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biol. significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants contg. both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.
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    12. 12
      Premkumar, L.; Segovia-Chumbez, B.; Jadi, R.; Martinez, D. R.; Raut, R.; Markmann, A. J.; Cornaby, C.; Bartelt, L.; Weiss, S.; Park, Y.; Edwards, C. E.; Weimer, E.; Scherer, E. M.; Rouphael, N.; Edupuganti, S.; Weiskopf, D.; Tse, L. V.; Hou, Y. J.; Margolis, D.; Sette, A.; Collins, M. H.; Schmitz, J.; Baric, R. S.; de Silva, A. M. The receptor-binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci. Immunol. 2020, 5, eabc8413  DOI: 10.1126/sciimmunol.abc8413
      There is no corresponding record for this reference.
    13. 13
      Pinto, D.; Park, Y.-J.; Beltramello, M.; Walls, A. C.; Tortorici, M. A.; Bianchi, S.; Jaconi, S.; Culap, K.; Zatta, F.; De Marco, A.; Peter, A.; Guarino, B.; Spreafico, R.; Cameroni, E.; Case, J. B.; Chen, R. E.; Havenar-Daughton, C.; Snell, G.; Telenti, A.; Virgin, H. W.; Lanzavecchia, A.; Diamond, M. S.; Fink, K.; Veesler, D.; Corti, D. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 2020, 583, 290– 295,  DOI: 10.1038/s41586-020-2349-y
      13
      Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody
      Pinto, Dora; Park, Young-Jun; Beltramello, Martina; Walls, Alexandra C.; Tortorici, M. Alejandra; Bianchi, Siro; Jaconi, Stefano; Culap, Katja; Zatta, Fabrizia; De Marco, Anna; Peter, Alessia; Guarino, Barbara; Spreafico, Roberto; Cameroni, Elisabetta; Case, James Brett; Chen, Rita E.; Havenar-Daughton, Colin; Snell, Gyorgy; Telenti, Amalio; Virgin, Herbert W.; Lanzavecchia, Antonio; Diamond, Michael S.; Fink, Katja; Veesler, David; Corti, Davide
      Nature (London, United Kingdom) (2020), 583 (7815), 290-295CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in >3.7 million infections and 260,000 deaths as of 6 May 2020. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. We describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope contg. a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails contg. S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.
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    14. 14
      Zhou, D.; Tian, X.; Qi, R.; Peng, C.; Zhang, W. Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics. Glycobiology 2020, 31, 69– 80,  DOI: 10.1093/glycob/cwaa052
      There is no corresponding record for this reference.
    15. 15
      Muhuri, M.; Gao, G. Is smaller better? Vaccine targeting recombinant receptor-binding domain might hold the key for mass production of effective prophylactics to fight the COVID-19 pandemic. Signal Transduction Targeted Ther. 2020, 5, 222  DOI: 10.1038/s41392-020-00317-1
      15
      Is smaller better? Vaccine targeting recombinant receptor-binding domain might hold the key for mass production of effective prophylactics to fight the COVID-19 pandemic
      Muhuri, Manish; Gao, Guangping
      Signal Transduction and Targeted Therapy (2020), 5 (1), 222CODEN: STTTCB; ISSN:2059-3635. (Nature Research)
      A review. A recent report by Yang et al. published in Nature (https://doi.org/10.1038/s41586-020-2599-8 (2020)) reported a recombinant vaccine utilizing recombinant receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein. This vaccine candidate successfully induced potent functional antibody responses in the immunized mice, rabbits, and non-human primates. The study highlights the crit. role of the immunogenicity of the RBD domain upon SARS-CoV-2 infection and the alternate vaccine designs that could serve as effective prophylactics against the pandemic.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVCmtLjE&md5=4fcbbfee5a90d6812953db01daf361ea
    16. 16
      He, Y.; Zhou, Y.; Wu, H.; Luo, B.; Chen, J.; Li, W.; Jiang, S. Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines. J. Immunol. 2004, 173, 4050– 4057,  DOI: 10.4049/jimmunol.173.6.4050
      16
      Identification of Immunodominant Sites on the Spike Protein of Severe Acute Respiratory Syndrome (SARS) Coronavirus: Implication for Developing SARS Diagnostics and Vaccines
      He, Yuxian; Zhou, Yusen; Wu, Hao; Luo, Baojun; Chen, Jingming; Li, Wanbo; Jiang, Shibo
      Journal of Immunology (2004), 173 (6), 4050-4057CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
      The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as detd. by Pepscan anal. with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S603-634, which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnsVWktLY%253D&md5=992029e2a2c37b44efa5d77f395ac64f
    17. 17
      Lenza, M. P.; Oyenarte, I.; Diercks, T.; Quintana, J. I.; Gimeno, A.; Coelho, H.; Diniz, A.; Peccati, F.; Delgado, S.; Bosch, A.; Valle, M.; Millet, O.; Abrescia, N. G. A.; Palazón, A.; Marcelo, F.; Jiménez-Osés, G.; Jiménez-Barbero, J.; Ardá, A.; Ereño-Orbea, J. Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins. Angew. Chem., Int. Ed. 2020, 59, 23763– 23771,  DOI: 10.1002/anie.202011015
      17
      Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins
      Lenza, Maria Pia; Oyenarte, Iker; Diercks, Tammo; Quintana, Jon Imanol; Gimeno, Ana; Coelho, Helena; Diniz, Ana; Peccati, Francesca; Delgado, Sandra; Bosch, Alexandre; Valle, Mikel; Millet, Oscar; Abrescia, Nicola G. A.; Palazon, Asis; Marcelo, Filipa; Jimenez-Oses, Gonzalo; Jimenez-Barbero, Jesus; Arda, Ana; Ereno-Orbea, June
      Angewandte Chemie, International Edition (2020), 59 (52), 23763-23771CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The glycan structures of the receptor-binding domain of the SARS-CoV-2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analyzed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD 13C-labeled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15N-labeled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary expts. from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.
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    18. 18
      Resemann, A.; Jabs, W.; Wiechmann, A.; Wagner, E.; Colas, O.; Evers, W.; Belau, E.; Vorwerg, L.; Evans, C.; Beck, A.; Suckau, D. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing. mAbs 2016, 8, 318– 330,  DOI: 10.1080/19420862.2015.1128607
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      Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing
      Resemann, Anja; Jabs, Wolfgang; Wiechmann, Anja; Wagner, Elsa; Colas, Olivier; Evers, Waltraud; Belau, Eckhard; Vorwerg, Lars; Evans, Catherine; Beck, Alain; Suckau, Detlev
      mAbs (2016), 8 (2), 318-330CODEN: MABSCP; ISSN:1942-0870. (Taylor & Francis, Inc.)
      The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the anal. data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called "bottom-up" approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall anal. time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their ref. sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced - the "Sequence Validation Percentage.". Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up mol. wt. detn. and middle-down protein sequencing. Three errors in the ref. amino acid sequence of natalizumab, causing a cumulative mass shift of only -2 Da in the natalizumab Fd domain, were cor. as a result of this work.
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    19. 19
      Gstöttner, C.; Reusch, D.; Haberger, M.; Dragan, I.; Van Veelen, P.; Kilgour, D. P. A.; Tsybin, Y. O.; van der Burgt, Y. E. M.; Wuhrer, M.; Nicolardi, S. Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry. mAbs 2020, 12, 1682403  DOI: 10.1080/19420862.2019.1682403
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      Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry
      Gstottner Christoph; Dragan Irina; Van Veelen Peter; van der Burgt Yuri E M; Wuhrer Manfred; Nicolardi Simone; Reusch Dietmar; Haberger Markus; Kilgour David P A; Tsybin Yury O
      mAbs (2020), 12 (1), 1682403 ISSN:.
      Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.
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    20. 20
      Santos, M. R.; Ratnayake, C. K.; Fonslow, B.; Guttman, A. A Covalent, Cationic Polymer Coating Method for the CESI-MS Analysis of Intact Proteins and Polypeptides, SCIEX Separations, Brea, CA, 2015.
      There is no corresponding record for this reference.
    21. 21
      Yang, Y.; Liu, F.; Franc, V.; Halim, L. A.; Schellekens, H.; Heck, A. J. R. Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity. Nat. Commun. 2016, 7, 13397  DOI: 10.1038/ncomms13397
      21
      Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity
      Yang, Yang; Liu, Fan; Franc, Vojtech; Halim, Liem Andhyk; Schellekens, Huub; Heck, Albert J. R.
      Nature Communications (2016), 7 (), 13397CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resoln. native mass spectrometry and middle-down proteomics, to analyze this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addn. unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quant. assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.
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    22. 22
      Hinneburg, H.; Stavenhagen, K.; Schweiger-Hufnagel, U.; Pengelley, S.; Jabs, W.; Seeberger, P. H.; Silva, D. V.; Wuhrer, M.; Kolarich, D. The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics. J. Am. Soc. Mass Spectrom. 2016, 27, 507– 519,  DOI: 10.1007/s13361-015-1308-6
      22
      The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics
      Hinneburg, Hannes; Stavenhagen, Kathrin; Schweiger-Hufnagel, Ulrike; Pengelley, Stuart; Jabs, Wolfgang; Seeberger, Peter H.; Silva, Daniel Varon; Wuhrer, Manfred; Kolarich, Daniel
      Journal of the American Society for Mass Spectrometry (2016), 27 (3), 507-519CODEN: JAMSEF; ISSN:1044-0305. (Springer)
      In-depth site-specific investigations of protein glycosylation are the basis for understanding the biol. function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable detn. of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied anal. tools, including glycopeptide fragmentation regimes and automated data anal. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissocn. (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS expt. We validated this method analyzing a set of human Igs (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compns., peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide anal. platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.
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    23. 23
      Zhang, T.; Madunić, K.; Holst, S.; Zhang, J.; Jin, C.; ten Dijke, P.; Karlsson, N. G.; Stavenhagen, K.; Wuhrer, M. Development of a 96-well plate sample preparation method for integrated N- and O-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry. Mol. Omics 2020, 16, 355– 363,  DOI: 10.1039/C9MO00180H
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      Development of a 96-well plate sample preparation method for integrated N- and O-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry
      Zhang, Tao; Madunic, Katarina; Holst, Stephanie; Zhang, Jing; Jin, Chunsheng; ten Dijke, Peter; Karlsson, Niclas G.; Stavenhagen, Kathrin; Wuhrer, Manfred
      Molecular Omics (2020), 16 (4), 355-363CODEN: MOOMAW ISSN:. (Royal Society of Chemistry)
      Changes in glycosylation signatures of cells have been assocd. with pathol. processes in cancer as well as infectious and autoimmune diseases. The current protocols for comprehensive anal. of N-glycomics and O-glycomics derived from cells and tissues often require a large amt. of biol. material. They also only allow the processing of very limited nos. of samples at a time. Here we established a workflow for sequential release of N-glycans and O-glycans based on PVDF membrane immobilization in 96-well format from 5 x 105 cells. Released glycans are reduced, desalted, purified, and reconstituted, all in 96-well format plates, without addnl. staining or derivatization. The developed protocol allows the anal. of N- and O-glycans from relatively large nos. of samples in a less time consuming way with high repeatability. Inter- and intraday repeatability of the fetuin N-glycan anal. showed two median intraday coeffs. of variations (CVs) of 7.6% and 8.0%, and a median interday CV of 9.8%. Median CVs of 7.9% and 8.7% for the main peaks of N- and O-glycans released from the NMuMG cell line indicate a very good repeatability. The method is applicable to purified glycoproteins as well as to biofluids and cell- or tissue-based samples.
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    24. 24
      Madunić, K.; Zhang, T.; Mayboroda, O. A.; Holst, S.; Stavenhagen, K.; Jin, C.; Karlsson, N. G.; Lageveen-Kammeijer, G. S. M.; Wuhrer, M. Colorectal cancer cell lines show striking diversity of their O-glycome reflecting the cellular differentiation phenotype. Cell. Mol. Life Sci. 2021, 78, 337– 350,  DOI: 10.1007/s00018-020-03504-z
      24
      Colorectal cancer cell lines show striking diversity of their O-glycome reflecting the cellular differentiation phenotype
      Madunic, Katarina; Zhang, Tao; Mayboroda, Oleg A.; Holst, Stephanie; Stavenhagen, Kathrin; Jin, Chunsheng; Karlsson, Niclas G.; Lageveen-Kammeijer, Guinevere S. M.; Wuhrer, Manfred
      Cellular and Molecular Life Sciences (2021), 78 (1), 337-350CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)
      Abstr.: Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their assocn. with other mol. features, an in-depth O-glycosylation anal. of 26 different CRC cell lines was performed. The released O-glycans were analyzed on porous graphitized carbon nano-liq. chromatog. system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC-ESI-MS/MS) allowing isomeric sepn. as well as in-depth structural characterization. Assocns. between the obsd. glycan phenotypes with previously reported cell line transcriptome signatures were examd. by canonical correlation anal. Striking differences are obsd. between the O-glycomes of 26 CRC cell lines. Unsupervized principal component anal. reveals a sepn. between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated α2,6-core sialylated glycans. Moreover, the expression of glycan signatures assocs. with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies.
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    25. 25
      Karlsson, N. G.; Wilson, N. L.; Wirth, H. J.; Dawes, P.; Joshi, H.; Packer, N. H. Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis. Rapid Commun. Mass Spectrom. 2004, 18, 2282– 2292,  DOI: 10.1002/rcm.1626
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      Negative ion graphitized carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis
      Karlsson, Niclas G.; Wilson, Nicole L.; Wirth, Hans-Juergen; Dawes, Peter; Joshi, Hiren; Packer, Nicolle H.
      Rapid Communications in Mass Spectrometry (2004), 18 (19), 2282-2292CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      Neg. ion nano-liq. chromatog./mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS2), using graphitized carbon as sepg. medium, were explored for analyzing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 μL/min) coupled with a conventional electrospray ionization source, the nano-LC/MS (flow rate of 0.6 μL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The abs. signals for the [M-2H]n- ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS2 mode, in which detection of low abundant sequence ions is necessary for characterization of highly sialylated N-linked oligosaccharides. Oligosaccharides with high nos. of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS2 anal. was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitized carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 μg of total amt. of protein).
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    26. 26
      Karlsson, N. G.; Schulz, B. L.; Packer, N. H. Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J. Am. Soc. Mass Spectrom. 2004, 15, 659– 672,  DOI: 10.1016/j.jasms.2004.01.002
      26
      Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn
      Karlsson, Niclas G.; Schulz, Benjamin L.; Packer, Nicolle H.
      Journal of the American Society for Mass Spectrometry (2004), 15 (5), 659-672CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
      Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were sepd. and detected by neg. ion LC-MS and LC-MS2. The resoln. of the chromatog. and the information obtained from collision induced dissocn. of detected [M - H]- ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nona-saccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Zi and Yi), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAcβ1-3GalNAcol or Gal/GlcNAcβ1-3(GlcNAcβ1-6)GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and Ai type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution (0,2Ai - H2O cleavage) and disubstitution of C-3 and C-4 (Zi/Zi cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Galβ1-3/4GlcNAcβ1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, neg. ion LC-MS2 was able to generate extensive data for understanding the overall glycosylation pattern of a sample, esp. when only a limited amt. of material is available.
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    27. 27
      Anugraham, M.; Everest-Dass, A. V.; Jacob, F.; Packer, N. H. A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells. Rapid Commun. Mass Spectrom. 2015, 29, 545– 561,  DOI: 10.1002/rcm.7130
      27
      A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells
      Anugraham, Merrina; Everest-Dass, Arun Vijay; Jacob, Francis; Packer, Nicolle H.
      Rapid Communications in Mass Spectrometry (2015), 29 (7), 545-561CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      Glycosphingolipids (GSLs) constitute a highly diverse class of glyco-conjugates which are involved in many aspects of cell membrane function and disease. The isolation, detection and structural characterization of the carbohydrate (glycan) component of GSLs are particularly challenging given their structural heterogeneity and thus rely on the development of sensitive, anal. technologies. Neutral and acidic GSL stds. were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymically released using endoglycoceramidase II (EGCase II), sepd. by porous graphitized carbon (PGC) liq. chromatog. and structurally characterized by neg. ion mode electrospray ionization tandem mass spectrometry (PGC-LC/ESI-MS/MS). This approach was then employed for GSLs isolated from 100 mg of serous and endometrioid cancer tissue and from cell line (107 cells) samples. Glycans were released from GSL stds. comprised of ganglio-, asialo-ganglio- and the relatively resistant globo-series glycans, using as little as 1 mU of enzyme and 2 μg of GSL. The platform of anal. was then applied to GSLs isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatog. resolved on PGC and characterized by comparison with the MS2 fragment ion spectra of the glycan stds. and by application of known structural MS2 fragment ions. This approach identified several (neo-)lacto-, globo- and ganglio-series glycans and facilitated the discrimination of isomeric structures contg. Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides. Thus, the authors describe a relatively simple, detergent-free, enzymic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural anal. afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the anal. of tumor and cell-derived GSL-glycans. The method uses the potential of MS2 fragmentation in neg. ion ESI mode to characterize, in detail, the biol. relevant glycan structures derived from GSLs.
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    28. 28
      Ceroni, A.; Maass, K.; Geyer, H.; Geyer, R.; Dell, A.; Haslam, S. M. GlycoWorkbench: A Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans. J. Proteome Res. 2008, 7, 1650– 1659,  DOI: 10.1021/pr7008252
      28
      GlycoWorkbench: A Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans
      Ceroni, Alessio; Maass, Kai; Geyer, Hildegard; Geyer, Rudolf; Dell, Anne; Haslam, Stuart M.
      Journal of Proteome Research (2008), 7 (4), 1650-1659CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Mass spectrometry is the main anal. technique currently used to address the challenges of glycomics as it offers unrivaled levels of sensitivity and the ability to handle complex mixts. of different glycan variations. Detn. of glycan structures from anal. of MS data is a major bottleneck in high-throughput glycomics projects, and robust solns. to this problem are of crit. importance. However, all the approaches currently available have inherent restrictions to the type of glycans they can identify, and none of them have proved to be a definitive tool for glycomics. GlycoWorkbench is a software tool developed by the EUROCarbDB initiative to assist the manual interpretation of MS data. The main task of GlycoWorkbench is to evaluate a set of structures proposed by the user by matching the corresponding theor. list of fragment masses against the list of peaks derived from the spectrum. The tool provides an easy to use graphical interface, a comprehensive and increasing set of structural constituents, an exhaustive collection of fragmentation types, and a broad list of annotation options. The aim of GlycoWorkbench is to offer complete support for the routine interpretation of MS data. The software is available for download from: http://www.eurocarbdb.org/applications/ms-tools.
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    29. 29
      Cooper, C. A.; Gasteiger, E.; Packer, N. H. GlycoMod--a software tool for determining glycosylation compositions from mass spectrometric data. Proteomics 2001, 1, 340– 349,  DOI: 10.1002/1615-9861(200102)1:2<340::AID-PROT340>3.0.CO;2-B
      29
      GlycoMod - A software tool for determining glycosylation compositions from mass spectrometric data
      Cooper, Catherine A.; Gasteiger, Elisabeth; Packer, Nicolle H.
      Proteomics (2001), 1 (2), 340-349CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH)
      GlycoMod is a software tool designed to find all possible compns. of a glycan structure from its exptl. detd. mass. The program can be used to predict the compn. of any glycoprotein-derived oligosaccharide comprised of either underivatized, methylated or acetylated monosaccharides, or with a derivatized reducing terminus. The compn. of a glycan attached to a peptide can be computed if the sequence or mass of the peptide is known. In addn., if the protein is known and is contained in the SWISS-PROT or TrEMBL databases, the program will match the exptl. detd. masses against all the predicted protease-produced peptides (including any post-translational modifications annotated in these databases) which have the potential to be glycosylated with either N- or O-linked oligosaccharides. Since many possible glycan compns. can be generated from the same mass, the program can apply compositional constraints to the output if the user supplies either known or suspected monosaccharide constituents. Furthermore, known oligosaccharide structural constraints on monosaccharide compn. are also incorporated into the program to limit the output.
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    30. 30
      Wohlschlager, T.; Scheffler, K.; Forstenlehner, I. C.; Skala, W.; Senn, S.; Damoc, E.; Holzmann, J.; Huber, C. G. Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals. Nat. Commun. 2018, 9, 1713  DOI: 10.1038/s41467-018-04061-7
      30
      Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals
      Wohlschlager Therese; Forstenlehner Ines C; Skala Wolfgang; Senn Stefan; Huber Christian G; Wohlschlager Therese; Scheffler Kai; Forstenlehner Ines C; Skala Wolfgang; Senn Stefan; Holzmann Johann; Huber Christian G; Scheffler Kai; Forstenlehner Ines C; Holzmann Johann; Damoc Eugen
      Nature communications (2018), 9 (1), 1713 ISSN:.
      Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom-up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process.
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    31. 31
      Hati, S.; Bhattacharyya, S. Impact of Thiol-Disulfide Balance on the Binding of Covid-19 Spike Protein with Angiotensin-Converting Enzyme 2 Receptor. ACS Omega 2020, 5, 16292– 16298,  DOI: 10.1021/acsomega.0c02125
      31
      Impact of Thiol-Disulfide Balance on the Binding of Covid-19 Spike Protein with Angiotensin-Converting Enzyme 2 Receptor
      Hati, Sanchita; Bhattacharyya, Sudeep
      ACS Omega (2020), 5 (26), 16292-16298CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an ongoing pandemic of coronavirus disease (COVID-19), which started in 2019. This is a member of Coronaviridae family in the genus Betacoronavirus, which also includes SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). The angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV and SARS-CoV-2 to enter the host cells. In particular, the interaction of viral spike proteins with ACE2 is a crit. step in the viral replication cycle. The receptor-binding domain of the viral spike proteins and ACE2 have several cysteine residues. In this study, the role of thiol-disulfide balance on the interactions between SARS-CoV/CoV-2 spike proteins and ACE2 was investigated using mol. dynamics simulations. The study revealed that the binding affinity was significantly impaired when all of the disulfide bonds of both ACE2 and SARS-CoV/CoV-2 spike proteins were reduced to thiol groups. The impact on the binding affinity was less severe when the disulfide bridges of only one of the binding partners were reduced to thiols. This computational finding possibly provides a mol. basis for the differential COVID-19 cellular recognition due to the oxidative stress.
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    32. 32
      Gstöttner, C.; Nicolardi, S.; Haberger, M.; Reusch, D.; Wuhrer, M.; Domínguez-Vega, E. Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS. Anal. Chim. Acta 2020, 1134, 18– 27,  DOI: 10.1016/j.aca.2020.07.069
      32
      Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS
      Gstottner, Christoph; Nicolardi, Simone; Haberger, Markus; Reusch, Dietmar; Wuhrer, Manfred; Dominguez-Vega, Elena
      Analytica Chimica Acta (2020), 1134 (), 18-27CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be obsd. contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new anal. strategies are needed for a more accurate and in-depth anal. Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a pos. coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the sepn. permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond redn. The six different subunits (Lc1, Lc2, Fd'1, Fd'2, (Fc/2)1 and (Fc/2)2) were sepd. using the same pos.-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resoln. (resolving power 140000 at m/z 1100) for a more confident anal. of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately detd. in the subunits showing the complementarity of both approaches.
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    33. 33
      Trastoy, B.; Naegeli, A.; Anso, I.; Sjögren, J.; Guerin, M. E. Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila. Nat. Commun. 2020, 11, 4844  DOI: 10.1038/s41467-020-18696-y
      33
      Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila
      Trastoy, Beatriz; Naegeli, Andreas; Anso, Itxaso; Sjogren, Jonathan; Guerin, Marcelo E.
      Nature Communications (2020), 11 (1), 4844CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the human gut that promotes a beneficial effect on health, likely based on the regulation of mucus thickness and gut barrier integrity, but also on the modulation of the immune system. In this work, we focus in OgpA from A. muciniphila, an O-glycopeptidase that exclusively hydrolyzes the peptide bond N-terminal to serine or threonine residues substituted with an O-glycan. We det. the high-resoln. X-ray crystal structures of the unliganded form of OgpA, the complex with the glycodrosocin O-glycopeptide substrate and its product, providing a comprehensive set of snapshots of the enzyme along the catalytic cycle. In combination with O-glycopeptide chem., enzyme kinetics, and computational methods we unveil the mol. mechanism of O-glycan recognition and specificity for OgpA. The data also contribute to understanding how A. muciniphila processes mucins in the gut, as well as anal. of post-translational O-glycosylation events in proteins.
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    34. 34
      Guile, G. R.; Harvey, D. J.; O’Donnell, N.; Powell, A. K.; Hunter, A. P.; Zamze, S.; Fernandes, D. L.; Dwek, R. A.; Wing, D. R. Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland. Eur. J. Biochem. 1998, 258, 623– 656,  DOI: 10.1046/j.1432-1327.1998.2580623.x
      34
      Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland
      Guile, Geoffrey R.; Harvey, David J.; O'Donnell, Niall; Powell, Andrew K.; Hunter, Ann P.; Zamze, Susanne; Fernandes, Daryl L.; Dwek, Raymond A.; Wing, David R.
      European Journal of Biochemistry (1998), 258 (2), 623-656CODEN: EJBCAI; ISSN:0014-2956. (Springer-Verlag)
      The glycosylation of a no. of constituents of human saliva is known to modify its biol. roles, such as its lubricating properties and binding of microbial flora. Gillece-Castro et al. [Gillece-Castro, B. L., Prakobphol, A., Burlingame, A. L., Leffler, H. & Fisher, S. J. (1991) J. Biol. Chem. 266, 17358-17368] have proposed that the major glycan on the salivary proline-rich glycoproteins is a trifucosylated biantennary sugar with one difucosylated and one unfucosylated antenna. Furthermore, they proposed that the non-fucosylated antenna mediated adherence to a periodontal pathogen, Fusobacterium nucleatum. The detailed structures and roles of other highly fucosylated glycans that co-exist in the parotid gland are not fully known. In view of the influence of outer-arm fucosylation on carbohydrate recognition processes in general, this paper reports the use of a combination of HPLC (normal and reversed phase), matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry and exoglycosidase digestions to dissect the detailed structures of the most abundant of these polyfucosylated glycans. For measurement of reversed-phase HPLC retention times, new calibration units were used which paralleled the glucose units used for normal-phase HPLC. These differed in that the difference in retention times were compared with those derived from a ladder of 2-aminobenzamide-labeled arabinose oligomers instead of the corresponding oligomers from partially hydrolyzed dextran. Over sixty neutral sugars were identified from the parotid gland and many of these were addnl. found substituted with sialic acid (both α2-3-linked and α2-6-linked) and sulfate. These glycans were mainly bi- and tri-antennary sugars with up to five and seven fucose residues resp., contg. fucose α1-3-linked to the outer-arm GlcNAc residues and α1-2-linked to the galactose. All fucosylated structures contained a core (α1-6-linked) fucose. The detailed structure of the trifucosylated biantennary glycan was confirmed, together with the structures of another 12 fucosylated biantennary glycans. Smaller amts. of hybrid and tetraantennary structures were also found and bisected glycans were shown to be constituents of parotid glycoproteins for the first time. Acidic glycans were mainly substituted with sialic acid. Most were monosialylated as the presence of fucose on the antennae was found to suppress the addn. of extra sialic acid moieties. The possible functional significance of highly fucosylated N-glycans is discussed in relation to their modification of the availability of other non-reducing terminal monosaccharides for recognition processes.
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    • Details on the Experimental Section; MALDI-ISD sequence coverage; deconvoluted mass spectra for the de-N-glycosylated, fucosidase-treated, and O-protease-treated intact RBDs; O-glycan site localization by MALDI-ISD; deconvoluted mass spectra for the galactosidase- and sialidase-treated RBDs; site-specific glycan-type distribution pie charts; results for the SARS-CoV-2 antibody binding assay; and relative quantification of O-released glycans and glycopeptides from RBDs (PDF)


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