Spatially-Resolved Exploration of the Mouse Brain Glycome by Tissue Glyco-Capture (TGC) and Nano-LC/MS (original) (raw)
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Glycans are diverse structured biomolecules that play crucial roles in various biological processes. Glycosylation, an enzymatic system through which various glycans are bound to proteins and lipids, is the most common and functionally crucial post-translational modification process. It is known to be associated with brain development, signal transduction, molecular trafficking, neurodegenerative disorders, psychopathologies, and brain cancers. Glycans in glycoproteins and glycolipids expressed in brain cells are involved in neuronal development, biological processes, and central nervous system maintenance. The composition and expression of glycans are known to change during those physiological processes. Therefore, imaging of glycans and the glycoconjugates in the brain regions has become a “hot” topic nowadays. Imaging techniques using lectins, antibodies, and chemical reporters are traditionally used for glycan detection. However, those techniques offer limited glycome detection....
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Glycans are among the most intriguing carriers of biological information in living systems. The structures of glycans not only convey the cells' physiological state, but also regulate cellular communication and responses by engaging receptors on neighboring cells and in the extracellular matrix. The assembly of simple monosaccharide building blocks into linear or branched oligo- and polysaccharides gives rise to a large repertoire of diverse glycan structures. Despite their structural complexity, individual glycans rarely engage their protein partners with high affinity. Yet, glycans modulate biological processes with exquisite selectivity and specificity. To correctly evaluate glycan interactions and their biological consequences, one needs to look beyond individual glycan structures and consider the entirety of the cell-surface landscape. There, glycans are presented on protein scaffolds, or are linked directly to membrane lipids, forming a complex, hierarchically organized ne...
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European Journal of Biochemistry, 1998
Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N-glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N-glycosylation occurring in neural tissue, we have characterised all of the major neutral N-linked oligosaccharides expressed in adult rat using a combination of matrix-assisted laser-desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal-phase HPLC, and two-dimensional HPLC mapping. Oligomannosidic glycans, Man (9Ϫ5)GlcNAc2, constituted approximately 15% of the total brain N-glycan pool. The other neutral N-glycan components consisted of a series of diantennary structures (6.5%), (2,6)branched triantennary glycans (1 %) and hybrid structures (3%). Both the complex and hybrid N-glycans were characterised by the presence of outer-arm A(1,3)-fucosylation (forming the Lewis x determinant), A(1,6)-core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N-glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man A(1,3)-arm. This isomeric form is indicative of the action of a novel β-hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N-linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.
Tools for Studying Glycans: Recent Advances in Chemoenzymatic Glycan Labeling
ACS Chemical Biology, 2017
The study of cellular glycosylation presents many challenges due, in large part, to the nontemplate driven nature of glycan biosynthesis and their structural complexity. Chemoenzymatic glycan labeling (CEGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CEGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CEGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CEGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.
Molecular & cellular proteomics : MCP, 2017
The intrinsic nature of glycosylation, namely non-template encoded, stepwise elongation and termination with a diverse range of isomeric glyco-epitopes (glycotopes), translates into ambiguity in most cases of mass spectrometry (MS)-based glycomic mapping. It is arguable that whether one needs to delineate every single glycomic entity, which may be counterproductive. Instead, one should focus on identifying as many structural features as possible that would collectively define the glycomic characteristics of a cell or tissue, and how these may change in response to self-programmed development, immuno-activation, and malignant transformation. We have been pursuing this line of analytical strategy that homes in on identifying the terminal sulfo-, sialyl and/or fucosylated glycotopes by comprehensive nanoLC-MS(2)-product dependent MS(3) analysis of permethylated glycans, in conjunction with development of a data mining computational tool, GlyPick, to enable an automated, high throughput...
Enrichment of glycopeptides for glycan structure and attachment site identification
Nature Methods, 2009
within or between different glycoproteins in a mixture. Capture of periodate-oxidized glycoproteins on hydrazide beads has previously been used for high-throughput mapping of protein Nlinked glycan sites . With this approach, formerly N-glycosylated peptides are released by PNGase F treatment, and then the glycosylation sites can be assayed. However, no information regarding glycan structures is made available, and PNGase F treatment works only for N-linked glycoproteins.
Journal of the American Society for Mass Spectrometry, 2018
Glycoconjugates are directly or indirectly involved in many biological processes. Due to their complex structures, the structural elucidation of glycans and the exploration of their role in biological systems have been challenging. Glycan pools generated through release from glycoprotein or glycolipid mixtures can often be very complex. For the sake of procedural simplicity, many glycan profiling studies choose to concentrate on a single class of glycoconjugates. In this paper, we demonstrate it feasible to cover glycosphingolipids, N-glycans, and O-glycans isolated from the same sample. Small volumes of human blood serum and ascites fluid as well as small mouse brain tissue samples are sufficient to profile sequentially glycans from all three classes of glycoconjugates and even positively identify some mixture components through MALDI-MS and LC-ESI-MS. The results show that comprehensive glycan profiles can be obtained from the equivalent of 500-μg protein starting material or poss...
Rapid Communications in Mass Spectrometry, 2015
RATIONALE: 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, analytical technologies. METHODS: Neutral and acidic GSL standards were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymatically released using endoglycoceramidase II (EGCase II), separated by porous graphitized carbon (PGC) liquid chromatography and structurally characterized by negative 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 (10 7 cells) samples. RESULTS: Glycans were released from GSL standards comprising 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 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. This approach identified several (neo-)lacto-, globo-and ganglio-series glycans and facilitated the discrimination of isomeric structures containing Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides. CONCLUSION: We describe a relatively simple, detergent-free, enzymatic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural analysis afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the analysis of tumour and cell-derived GSL-glycans. The method uses the potential of MS 2 fragmentation in negative ion ESI mode to characterize, in detail, the biologically relevant glycan structures derived from GSLs.