All of the fragment ions of the glycan chain were assigned to known glycan structures. Nlinked glycans are attached in the endoplasmic reticulum to the nitrogen n in the side chain of asparagine in the sequon. Structural analysis of the nlinked glycan chains from a stylar glycoprotein associated with expression of selfincompatibility in nicotiana alata. Ludgers scientists have extensive experience in detailed glycan analysis and their expertise can be applied to both biologic drugs biopharmaceuticals and glycoconjugates relevant to medical research. Tentatively identify individual oligosaccharide species. Only a few nanograms of each glycan is linked to the slide in a spot diameter of. The nlinked glycans of fc are typically complextype, forming a network of noncovalent interactions along the protein surface of the c. Structural analysis of n and oglycans released from. Structural analysis of glycans in accordance with ich guidelines glycosylation is a dynamic, biologically active posttranslational modification factors affecting glycosylation include the cellline and bioreactor conditions which may produce the same protein backbone with differences in overall structure, linkage and composition of. The emergence of the betacoronavirus, sarscov2, the causative agent of covid19, represents a significant threat to global human health.
Occurrence and structural analysis of highly sulfated multiantennary nlinked glycan chains derived from a fertilizationassociated carbohydraterich glycoprotein in unfertilized eggs of tribolodon hakonensis. However, when an analyte contains many residues of the same mass, as in the case of glycans, it is sometimes difficult to determine the origin of the fragment ions from the glycan. This chapter surveys methods for the structural characterization of glycans, including residue. Sarscov2 s gene encodes 22 nlinked glycan sequons per protomer, which likely play a role in immune. Sitespecific glycan analysis of the sarscov2 spike. The platform of analysis was then applied to gsls isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatographically resolved on pgc and characterized by comparison with the ms 2 fragment ion spectra of the glycan standards and by application of known structural ms 2 fragment ions. The next generation of glycan sample preparation and analysis.
Glycan analysis therefore requires highresolution separation techniques that can provide detailed structural analysis, including both monosaccharide sequence and linkage information. Glycan id monoisotopic mass glycan structure % rsd based on 16 samples run highresolution glycan identification and quantitation in minutes run your glycans in record time glycan analysis has traditionally required a lengthy sample preparation process, a long. However, both technologies are not universal enough and have. The bioaccord system with smartms brings comprehensive nlinked glycan analysis to a new level. An example of glycan reducing sugar analysis and the corresponding glycan alditol analysis can be done and is shown below 46. Structural biochemistrycarbohydratesglycans wikibooks. A comprehensive approach for monoclonal antibody nlinked. Mass spectrometry methods are used for mapping glycoprotein glycosylation and detailed glycan structural determination. Structural analysis of the nlinked glycan chains from a. Normal phase, weak anion exchange, and reversed phase chromatography are most commonly used in conjunction with fluorescentlabeled oligosaccharides for the. Released nlinked glycan analysis using the bioaccord system. Characterization of glycoproteins routinely involves glycan analysis to monitor. However, coelution of different charge state glycans.
Waters offers complete, workflowbased approaches for analyzing glycoproteins at all structural levels. Glycosylation of proteins structure, function and analysis. Conclusions glycan analysis using uhplcfldqtof ms on the agilent 1290 infinity lc system and the agilent 6520 qtof lcms system is a powerful platform for quantitative and qualitative glycan profiling. Structural analysis of glycans essentials of glycobiology ncbi. It is therefore essential to separate all isomeric, charge, and branching glycan variations to understand the detailed structure of the glycans by lcmsms methods. Glycan analysis with hpaepad workflow for glycan analysis using hpaepad glycan analysis although determining monosaccharide content in glycoproteins provides significant information, knowledge of glycan structure is valuable. First, regulatory authorities were beginning to scrutinize the glycan structures of biopharmaceutical products more carefully based on established technical guidelines e. Glycomics is the comprehensive study of glycomes the entire complement of sugars, whether free or present in more complex molecules of an organism, including genetic, physiologic, pathologic, and other aspects. Glycan analysis glycan analysis by ion mobilitymass.
Glycan analysis enables monitoring of relative quantities of a particular set of glycans. Protein glycosylation, the most universal and diverse posttranslational modification, can affect protein secretion, stability, and immunogenicity. Indepth structural analysis of glycans in the gas phase eike mucha,ab alexandra stuckmann,ab mateusz marianski,a weston b. Human igg fc glycosylation modulates immunological effector functions such as antibodydependent cellular cytotoxicity and phagocytosis. In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants. The built glycosylation shield structures of covid19 and sarscov spike glycoproteins were superimposed and are shown in figure 1c.
Effective use of mass spectrometry for glycan and glycopeptide structural analysis most proteins are glycosylated. Although several analytical techniques are used for glycan structural analysis, mass spectrometry occupies an important. Primarily, separation can reduce sample complexity, minimize ion suppression, increase dynamic range of analysis and provide separation of structural isomers. Occurrence and structural analysis of highly sulfated multiantennary nlinked glycan chains derived from a fertilizationassociated carbohydraterich glycoprotein in unfertilized eggs. Just as is observed with proteins in nature, additional structural diversity can be imparted to glycans by modifying their hydroxy groups with phosphate, sulfate, or acetyl esters.
A representative example of the analysis is shown in figure 3. The detailed structural information obtained from the msms data further validated the ability of the glycanpac axh1 column to separate glycans based on charge, size, and polarity. Structural analysis of glycans in accordance with ich guidelines same protein backbone with differences in overall structure, linkage and composition of glycans, potentially affecting bioactivity, safety and efficacy of the final product characterization of glycan structures is important from early stage cell line selection right. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. Glycan id monoisotopic mass glycan structure % rsd based on 16 samples run highresolution glycan identification and quantitation in minutes run your glycans in record time glycan analysis has traditionally required a lengthy sample preparation process, a long separation, and timeconsuming data analysis.
In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining. Sitespecific analysis of the sarscov2 glycan shield. Structural analysis of n and oglycans released from glycoproteins article pdf available in nature protocol 77. Although there have been substantial improvements in glycan analysis over the past decade, the lack of both highresolution and highthroughput methods hampers progress in glycomics. Structural basis of glycan diversity essentials of. Fast glycan labeling and analysis kit application guide. Sitespecific analysis of the sarscov2 glycan shield biorxiv. The first printed glycan microarray, available in 2005, was v2.
Analysis of nlinked glycan using maldimini1 compact maldi digital ion trap mass spectrometer. The primary structure of a glycan is defined not only by the nature and order of constituent monosaccharides, but also by the configuration and position of glycosidic linkages and the nature and location of nonglycan substituents see chapter 2. Pdf structural analysis of n and oglycans released from. Sarscov2 s gene encodes 22 nlinked glycan sequons per protomer, which likely play a role in. Guide to glycan analysis maximize glycan structural information monosaccharide analysis glycan analysis glycosylation site pro ling intact glycoprotein pro ling. Perform relative quantitation for individual species on a mole percent basis by integrating the absorbance signal from the chromophore label. This technique is nondestructive and can provide full structural information about the glycan being analyzed. There is the possibility of ion coisolation leading to incorrect interpretations. Glycan functions are often dependent on the structure of the oligosaccharide.
Detailed glycan structural information is crucial to further understand disease and to find vaccine candidates. Chemical and structural analysis of an antibody folding. The combination of the positively charged instantpc tag and sensitive agilent jet stream ajs electrospray. Structural analysis of glycans in accordance with ich guidelines same protein backbone with differences in overall structure, linkage and composition of glycans, potentially affecting bioactivity, safety and efficacy of the final product characterization of glycan structures is. Additionally it can provide valuable information regarding changes in glycan patterns or identify aberrant glycans. Because this method is nondestructive, the same sample can later be used for other, destructive approaches e. Nglycan structural similarity including their orientations with respect to the protein is discussed separately below. Compared to proteomics the mass spectrometric glycan analysis still employs a lot of manual work and the differential glycomics can be a. Toestablish the method, many investigations were focused on synthetic compounds or small biologically relevant carbohydrates for which prior structural knowledge existed. The fast glycan labeling and analysis kit is for laboratory use onlyk introduction the protocol consists of the following steps.
Glycan release and derivatizationthe structural analysis was performed on of. Pdf structural analysis of n and oglycans released. Especially for larger glycans, the analysis of specific fragments can be more informative than the analysis of intact isomeric precursor ions. Glycomics is the systematic study of all glycan structures of a given cell type or organism and is a subset of glycobiology. Structural analysis of glycans essentials of glycobiology. We have previously validated our glycopeptide analysis methodology and applied this approach to the study of a range of other viral glycoprotein immunogens4,2023, which enables crossviral comparisons of glycosylation to be made. Glycan may also refer to carbohydrate portions of glycoprotein, glycolipid, or a proteoglycan.
Structural analysis of n and o glycans released from glycoproteins, nat. Analysis of nglycans is assisted by the existence of several enzymes that can release nglycans. Waters offers complete, workflow based approaches for analyzing glycoproteins at all structural levels. Although several analytical techniques are used for glycan structural analysis, mass spectrometry occupies an important position due to its high sensitivity and selectivity. This perspective article highlights the current developments of liquid chromatography, mass spectrometry, ionmobility spectro most popular 20182019 analytical chemistry articles. The former suffers from its low efficiency while the latter two suffer from the possibility of missing the actual glycan structures. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural.
The built glycosylation shield structures of covid19 and sarscov spike glycoproteins were superimposed and are. Nglycan profiling analysis of a monoclonal antibody using. Vaccine development is focused on the principal target of the humoral immune response, the spike s glycoprotein, which mediates cell entry and membrane fusion. Though glycan analysis can be done by direct infusion into the mass spectrometer, separation prior to ms analysis can provide number of benefits. Glycan analysis solutions brochure waters corporation. The emergence of the betacoronavirus, sarscov2 that causes covid19, represents a significant threat to global human health. Structural analysis of labeled nglycans from proteins by. In the case of many mab nglycans, this composition is sufficient to achieve a highconfidence structural assignment. The sequon is an asnxser or asnxthr sequence, where x is any amino acid except proline and the glycan may be composed of nacetylgalactosamine, galactose, neuraminic acid, nacetylglucosamine, fucose, mannose, and other monosaccharides. Typically, the analysis of glycolipid glycans by nmr or ms is preceded by their purification using. Figure 2 shows the rmsd distributions of the nglycan structure pairs in the pdb and random conformation pool. Request pdf structural analysis of glycans analytical and detection methods glycoprotein, glycolipid, structural characterization find. Another highly promising approach to disentangle the complex structure of carbohydrates is a fragmentbased analysis using imms.
However, as with all tandem ms methods, confidence in the interpretation depends on clear understanding of the limitations of the method. Enzymatic release of the nglycans from glycoproteins. In summary, our study pinpointed, for the first time, that the pattern and level. Sep 12, 2016 the map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. Sitespecific glycan analysis of the sarscov2 spike science. Structural analysis and identification of sialyl linkage isomers in recent years, mass spectroscopy ms has been widely used in glycan analysis. The applied biosystems glycanassure hyperformance apts system is a rapid nlinked glycan release, labeling, and clean up system that provides flexibility in sample preparation and in glycan analysis while ensuring accurate and consistent data through complete deglycosylation and preservation of sialic acid. Indepth structural analysis of glycans in the gas phase.
Structural analysis of variable domain glycosylation of anti. We also performed the same analysis for the sarscov spike protein, to identify signi. Hplc analysis of glycans the glycan pool released from a glycoprotein can be separated into components depending upon the characteristics of the glycans and the hplc matrices used. Of these, the most wellknown is peptidenglycosidase f pngase f, an amidase that releases intact complex bianennary, highmannose and hybridtype nglycans by cleaving between the innermost glcnac of the nglycan and asparagine, unless the glcnac is. Structural analysis of n and oglycans released from glycoproteins. Themost obvious application of imms is the analysis of intact glycan ions,either for synthesis control or for structural identifications.
Pdf this protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it. This paper describes the purification and characterization of the nlinked glycan chains from these two glycoproteins. Comprehensive nglycan profiling, characterization and detailed. Glycan structural analysis by msn analysis in the ms2 glycan analysis, fragment ions caused by the cleavage of glycosyl bonds was mainly detected. Pdf indepth structural analysis of glycans in the gas phase. Structural analysis of glycans analytical and detection methods. Struwe, c gerard meijera and kevin pagelab although there have been substantial improvements in glycan analysis over the past decade, the lack of both highresolution and highthroughput methods hampers progress. Glycans can be homo or heteropolymers of monosaccharide residues. Earlier studies have indicated that the single potential nglycosylation site on the s 1glycoprotein bears a glycan chain, whereas of the four potential nglycosylation sites on the s 2glycoprotein, three are glycosylated. Glycan shield and epitope masking of a coronavirus spike. Mar 16, 2015 the platform of analysis was then applied to gsls isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatographically resolved on pgc and characterized by comparison with the ms 2 fragment ion spectra of the glycan standards and by application of known structural ms 2 fragment ions.
The incident was a watershed moment in the biopharmaceutical industry, marking the emergence of new challenges 1. Assess the complexity of the oligosaccharide mixture present on the protein. Complete structural elucidation requires full assignment of both the 1 h and cnmr. Mass spectrometry ms provides key structural information and has been extensively used as an orthogonal analytical technique for glycan characterization in biotherapeutics development. Novel glycan column technology for the lcms analysis of. Due to the complexity of a glycan structure, structural elucidation. Glycosylation is a prevalent posttranslational modification playing significant roles in cellular interactions and changing with the onset of cancer. Fast glycan labeling and analysis kit for the ma m plus pharmaceutical analysis system application guide.
Restricted nglycan conformational space in the pdb and. A platform for the structural characterization of glycans. Comprehensive nglycan profiling, characterization and. Complete structural elucidation requires full assignment of both the 1 h. Mar 28, 2020 the emergence of the betacoronavirus, sarscov2 that causes covid19, represents a significant threat to global human health. Orbitrap fusion for glycan and glycopeptide analysis. Magnetic beadmediated capture of the released glycans. Each glycan target is printed at the same concentration 100. For a typical mammalian glycoprotein, the aim is often to identify the correct structure from a range of known or predictable candidate. This approach is clearly the most powerful mass spectrometric method for glycan structural analysis. Nmr analysis of glycans nuclear magnetic resonance nmr spectroscopy measures the extent to which a glycan or other molecule distorts a magnetic field. Engineering of fc glycans therefore enables finetuning of the therapeutic properties of monoclonal antibodies. Pdf occurrence and structural analysis of highly sulfated.