Mass spectrometry-based shotgun glycomics for discovery of natural ligands of glycan-binding proteins

The non-covalent associations of complex carbohydrates (glycans) with glycan-binding proteins mediate many important physiological and pathophysiological processes. Identifying these interactions is essential to understanding their diverse biological functions and enables the development of new disease treatments and diagnostics. Knowledge of the repertoire of glycans recognized by most glycan-binding proteins and their affinities is incomplete. Mass spectrometry-based screening of natural glycan libraries has emerged as a promising approach to defining the glycan interactome of glycan-binding proteins. Here, we review recent advances in mass spectrometry-based natural library screening that have led to the discovery of glycan ligands of endogenous and exogenous proteins and illuminated their binding specificities.     

Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays

DNA and protein arrays are commonly accepted as powerful exploratory tools in research. This has mainly been achieved by the establishment of proper guidelines for quality control, allowing cross-comparison between different array platforms. As a natural extension, glycan microarrays were subsequently developed, and recent advances using such arrays have greatly enhanced our understanding of protein-glycan recognition in nature. However, although it is assumed that biologically significant protein-glycan binding is robustly detected by glycan microarrays, there are wide variations in the methods used to produce, present, couple, and detect glycans, and systematic cross-comparisons are lacking. We address these issues by comparing two arrays that together represent the marked diversity of sialic acid modifications, linkages, and underlying glycans in nature, including some identical motifs. We compare and contrast binding interactions with various known and novel plant, vertebrate, and viral sialic acid-recognizing proteins and present a technical advance for assessing specificity using mild periodate oxidation of the sialic acid chain. These data demonstrate both the diversity of sialic acids and the analytical power of glycan arrays, showing that different presentations in different formats provide useful and complementary interpretations of glycan-binding protein specificity. They also highlight important challenges and questions for the future of glycan array technology and suggest that glycan arrays with similar glycan structures cannot be simply assumed to give similar results.     

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.     

Annotation of a serum N-glycan library for rapid identification of structures

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible because of the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC-MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples.     

Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays

Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.     

Hexapeptide library as a universal tool for sample preparation in protein glycosylation analysis

Recent analytical advancements allow for large-scale glycomics and glycan-biomarker research with N-glycans released from complex protein mixtures of e.g. plasma with a wide range of protein concentrations. Protein enrichment techniques to obtain samples with a better representation of low-abundance proteins are hardy applied. In this study, hexapeptide ligands previously described for enrichment of low-abundance proteins in proteomics are evaluated for glycan analysis. A repeatable on-bead glycan release strategy was developed, and glycans were analyzed using capillary sieving electrophoresis on a DNA analyzer. Binding of proteins to the hexapeptide library occurred via the protein backbone. At neutral pH no discrimination between protein glycoforms was observed. Interestingly, glycan profiles of plasma with and without hexapeptide library enrichment revealed very similar patterns, despite the vast changes in protein concentrations in the samples. The most significant differences in glycosylation profiles were ascribed to a reduction in immunoglobulin-derived glycans. These results suggest that specific and sensitive biomarkers will be hard to access on the full plasma level using protein enrichment in combination with glycan analysis. Instead, fractionation techniques or profiling strategies on the glycopeptide level after enrichment are proposed for in-depth glycoproteomics research.     

Glycomic Analysis of Human Respiratory Tract Tissues and Correlation with Influenza Virus Infection

The first step in influenza infection of the human respiratory tract is binding of the virus to sialic (Sia) acid terminated receptors. The binding of different strains of virus for the receptor is determined by the α linkage of the sialic acid to galactose and the adjacent glycan structure. In this study the N- and O-glycan composition of the human lung, bronchus and nasopharynx was characterized by mass spectrometry. Analysis showed that there was a wide spectrum of both Sia α2-3 and α2-6 glycans in the lung and bronchus. This glycan structural data was then utilized in combination with binding data from 4 of the published glycan arrays to assess whether these current glycan arrays were able to predict replication of human, avian and swine viruses in human ex vivo respiratory tract tissues. The most comprehensive array from the Consortium for Functional Glycomics contained the greatest diversity of sialylated glycans, but was not predictive of productive replication in the bronchus and lung. Our findings indicate that more comprehensive but focused arrays need to be developed to investigate influenza virus binding in an assessment of newly emerging influenza viruses.     

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.², describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

Glycomics: an integrated systems approach to structure-function relationships of glycans

In comparison with genomics and proteomics, the advancement of glycomics has faced unique challenges in the pursuit of developing analytical and biochemical tools and biological readouts to investigate glycan structure-function relationships. Glycans are more diverse in terms of chemical structure and information density than are DNA and proteins. This diversity arises from glycans' complex nontemplate-based biosynthesis, which involves several enzymes and isoforms of these enzymes. Consequently, glycans are expressed as an 'ensemble' of structures that mediate function. Moreover, unlike protein-protein interactions, which can be generally viewed as 'digital' in regulating function, glycan-protein interactions impinge on biological functions in a more 'analog' fashion that can in turn 'fine-tune' a biological response. This fine-tuning by glycans is achieved through the graded affinity, avidity and multivalency of their interactions. Given the importance of glycomics, this review focuses on areas of technologies and the importance of developing a bioinformatics platform to integrate the diverse datasets generated using the different technologies to allow a systems approach to glycan structure-function relationships.     

Glycoscience aids in biomarker discovery

The glycome consists of all glycans (or carbohydrates) within a biological system, and modulates a wide range of important biological activities, from protein folding to cellular communications. The mining of the glycome for disease markers represents a new paradigm for biomarker discovery; however, this effort is severely complicated by the vast complexity and structural diversity of glycans. This review summarizes recent developments in analytical technology and methodology as applied to the fields of glycomics and glycoproteomics. Mass spectrometric strategies for glycan compositional profiling are described, as are potential refinements which allow structure-specific profiling. Analytical methods that can discern protein glycosylation at a specific site of modification are also discussed in detail. Biomarker discovery applications are shown at each level of analysis, highlighting the key role that glycoscience can play in helping scientists understand disease biology.     

A lectin microarray approach for the rapid analysis of bacterial glycans

Rapid evaluation of microbial cell-surface carbohydrates is essential to understanding the mechanisms by which bacteria use glycans to establish pathogenic or symbiotic relationships. Microbial glycan analysis is complicated both by the vast diversity of possible carbohydrate structures and by their dynamic nature. Bacteria can rapidly alter their glycan coats by switching the genes that are involved on and off in a phase-variable manner. Currently, there is a lack of appropriate tools for studying dynamic carbohydrate alterations. Here, we present a lectin microarray protocol for the high-throughput evaluation of cell-surface microbial sugars. The binding patterns of fluorescent bacteria to these arrays provide a simple means to fingerprint bacteria based on their surface carbohydrates. In addition, this method provides a rapid, parallel evaluation of glycans from multiple bacterial samples, allowing dynamic changes in carbohydrate structures to be studied. The entire procedure takes approximately 12 h but the printing of the microarray can be performed in advance.     

Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

All currently identified primary receptors of adeno-associated virus (AAV) are glycans. Depending on the AAV serotype, these carbohydrates range from heparan sulfate proteoglycans (HSPG), through glycans with terminal α2-3 or α2-6 sialic acids, to terminal galactose moieties. Receptor identification has largely relied on binding to natural compounds, defined glycan-presenting cell lines, or enzyme-mediated glycan modifications. Here, we describe a comparative binding analysis of highly purified, fluorescent-dye-labeled AAV vectors of various serotypes on arrays displaying over 600 different glycans and on a specialized array with natural and synthetic heparins. Few glycans bind AAV specifically in a serotype-dependent manner. Differential glycan binding was detected for the described sialic acid-binding AAV serotypes 1, 6, 5, and 4. The natural heparin binding serotypes AAV2, -3, -6, and -13 displayed differential binding to selected synthetic heparins. AAV7, -8, -rh.10, and -12 did not bind to any of the glycans present on the arrays. For discrimination of AAV serotypes 1 to 6 and 13, minimal binding moieties are identified. This is the first study to differentiate the natural mixed heparin binding AAV serotypes 2, 3, 6, and 13 by differential binding to specific synthetic heparins. Also, sialic acid binding AAVs display differential glycan binding specificities. The findings are relevant for further dissection of AAV host cell interaction. Moreover, the definition of single AAV-discriminating glycan binders opens the possibility for glycan microarray-based discrimination of AAV serotypes in gene therapy.     

Sweet spots in functional glycomics

Information contained in the mammalian glycome is decoded by glycan-binding proteins (GBPs) that mediate diverse functions including host-pathogen interactions, cell trafficking and transmembrane signaling. Although information on the biological roles of GBPs is rapidly expanding, challenges remain in identifying the glycan ligands and their impact on GBP function. Protein-glycan interactions are typically low affinity, requiring multivalent interactions to achieve a biological effect. Though many glycoproteins can carry the glycan structure recognized by the GBP, other factors, such as recognition of protein epitopes and microdomain localization, may restrict which glycoproteins are functional ligands in situ. Recent advances in development of glycan arrays, synthesis of multivalent glycan ligands, bioengineering of cell-surface glycans and glycomics databases are providing new tools to identify the ligands of GBPs and to elucidate the mechanisms by which they participate in GBP function.     

The N’s and O’s of Drosophila glycoprotein glycobiology

The past 25 years have seen significant advances in understanding the diversity and functions of glycoprotein glycans in Drosophila melanogaster. Genetic screens have captured mutations that reveal important biological activities modulated by glycans, including protein folding and trafficking, as well as cell signaling, tissue morphogenesis, fertility, and viability. Many of these glycan functions have parallels in vertebrate development and disease, providing increasing opportunities to dissect pathologic mechanisms using Drosophila genetics. Advances in the sensitivity of structural analytic techniques have allowed the glycan profiles of wild-type and mutant tissues to be assessed, revealing novel glycan structures that may be functionally analogous to vertebrate glycans. This review describes a selected set of recent advances in understanding the functions of N-linked and O-linked (non-glycosaminoglycan) glycoprotein glycans in Drosophila with emphasis on their relatedness to vertebrate organisms.     

Prospects for glycoinformatics

High-throughput and automated techniques (mass spectrometry in particular) allow increasingly rapid structural analysis of complex glycans. Information concerning the primary structure (composition, sequence and linkages), three-dimensional structure (including dynamics) and interactions of glycans is now available in sufficient quantity to justify the maintenance of databases and search facilities. Several such resources (both commercial and open access) are now available as web tools. To derive the full value of glycan databases, it will be necessary to develop a universally accepted machine-readable structural representation of glycans.     

Reductive Alkaline Release of N‐Glycans Generates a Variety of Unexpected,Useful Products

Release of O‐glycans by reductive β‐elimination has become routine in many glyco‐analytical laboratories and concomitant release of N‐glycans has repeatedly been observed. Revisiting this somewhat forgotten mode of N‐glycan release revealed that all kinds of N‐glycans including oligomannosidic and complex‐type N‐glycans from plants with 3‐linked fucose and from mammals with or without 6‐linked fucose and with sialic acid could be recovered. However, the mass spectra of the obtained products revealed very surprising facts. Even after 16 h incubation in 1 M sodium borohydride, a large part of the glycans occurred in reducing form. Moreover, about one third emerged in the form of the stable amino‐functionalized 1‐amino‐1‐deoxy‐glycitol. When avoiding acidic conditions, considerable amounts of glycosylamine were observed. In addition, a compound with a reduced asparagine and de‐N‐acetylation products, in particular of sialylated glycans, was seen. The relative yields of the products reducing glycosylamine, reducing N‐glycan, 1‐amino‐1‐deoxy‐glycitol or glycitol could be controlled by the release conditions, foremost by temperature and borohydride concentration. Thus, chemical release of N‐glycans constitutes a cost‐saving alternative to enzymatic hydrolysis for the preparation of precursors for the production of reference compounds for various formats of N‐glycan analysis. Moreover, it allows to obtain a stable amino‐functionalized glycan derivative, which can be employed to construct glycan arrays or affinity matrices.     

Evolutionary considerations in relating oligosaccharide diversity to biological function.

The oligosaccharide chains (glycans) attached to cell surface and extracellular proteins and lipids are known to mediate many important biological roles. However, for many glycans, there are still no evident functions that are of obvious benefit to the organism that synthesizes them. There is also no clear explanation for the extreme complexity and diversity of glycans that can be found on a given glycoconjugate or cell type. Based on the limited information available about the scope and distribution of this diversity among taxonomic groups, it is difficult to see clear trends or patterns consistent with different evolutionary lineages. It appears that closely related species may not necessarily share close similarities in their glycan diversity, and that more derived species may have simpler as well as more complex structures. Intraspecies diversity can also be quite extensive, often without obvious functional relevance. We suggest one general explanation for these observations, that glycan diversification in complex multicellular organisms is driven by evolutionary selection pressures of both endogenous and exogenous origin. We argue that exogenous selection pressures mediated by viral and microbial pathogens and parasites that recognize glycans have played a more prominent role, favoring intra- and interspecies diversity. This also makes it difficult to appreciate and elucidate the specific endogenous roles of the glycans within the organism that synthesizes them.     

Versatile fluorescent derivatization of glycans for glycomic analysis

The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.