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The structural characterization of glycans by mass spectrometry is particularly challenging. This is because of the high degree of isomerism in which glycans of the same mass can differ in their stereochemistry, attachment points, and degree of branching. Here we show that the addition of cryogenic vibrational spectroscopy to mass and mobility measurements allows one to uniquely identify and characterize these complex biopolymers. We investigate six disaccharide isomers that differ in their stereochemistry, attachment point of the glycosidic bond, and monosaccharide content, and demonstrate that we can identify each one unambiguously. Even disaccharides that differ by a single stereogenic center or in the monosaccharide sequence order show distinct vibrational fingerprints that would clearly allow their identification in a mixture, which is not possible by ion mobility spectrometry/mass spectrometry alone. Moreover, this technique can be applied to larger glycans, which we demonstrate by distinguishing isomeric branched and linear pentasaccharides. The creation of a database containing mass, collision cross section, and vibrational fingerprint measurements for glycan standards should allow unambiguous identification and characterization of these biopolymers in mixtures, providing an enabling technology for all fields of glycoscience. Graphical Abstract ᅟ

Ageing is a complex process which implies the accumulation of molecular, cellular and organ damage, leading to an increased vulnerability to disease. In Western societies, the increase in the elderly population, which is accompanied by ageing-associated pathologies such as cardiovascular and mental diseases, is becoming an increasing economic and social burden for governments. In order to prevent, treat and determine which subjects are more likely to develop these age-related diseases, predictive biomarkers are required. In this sense, some studies suggest that glycans have a potential role as disease biomarkers, as they modify the functions of proteins and take part in intra- and intercellular biological processes. As the glycome reflects the real-time status of these interactions, its characterisation can provide potential diagnostic and prognostic biomarkers for multifactorial diseases. This review gathers the alterations in protein glycosylation profiles that are associated with ageing and age-related diseases, such as cancer, type 2 diabetes mellitus, metabolic syndrome and several chronic inflammatory diseases. Furthermore, the review includes the available techniques for the determination and characterisation of glycans, such as liquid chromatography, electrophoresis, nuclear magnetic resonance and mass spectrometry.

The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.

Monoclonal antibodies (mAbs) represent the largest class of therapeutic protein drug products. mAb glycosylation produces a heterogeneous, analytically challenging distribution of glycoforms that typically should be adequately characterized because glycosylation-based product quality attributes (PQAs) can impact product quality, immunogenicity, and efficacy. In this study, two products were compared using a panel of analytical methods. Two high-resolution mass spectrometry (HRMS) workflows were used to analyze N-glycans, while nuclear magnetic resonance (NMR) was used to generate monosaccharide fingerprints. These state-of-the-art techniques were compared to conventional analysis using hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection (FLD). The advantages and disadvantages of each method are discussed along with a comparison of the identified glycan distributions. The results demonstrated agreement across all methods for major glycoforms, demonstrating how confidence in glycan characterization is increased by combining orthogonal analytical methodologies. The full panel of methods used represents a diverse toolbox that can be selected from based on the needs for a specific product or analysis.

The structural analysis of glycoproteins is a challenging endeavor and is under steadily increasing demand, but only a very limited number of labs have the expertise required to accomplish this task. This tutorial is aimed at researchers from the fields of molecular biology and biochemistry that have discovered that glycoproteins are important in their biological research and are looking for the tools to elucidate their structure. It provides brief descriptions of the major and most common analytical techniques used in glycomics and glycoproteomics analysis, including explanations of the rationales for individual steps and references to published literature containing the experimental details necessary to carry out the analyses. Glycomics includes the comprehensive study of the structure and function of the glycans expressed in a given cell or organism along with identification of all the genes that encode glycoproteins and glycosyltransferases. Glycoproteomics which is subset of both glycomics and proteomics is the identification and characterization of proteins bearing carbohydrates as posttranslational modification. This tutorial is designed to ease entry into the glycomics and glycoproteomics field for those without prior carbohydrate analysis experience.

Mass spectrometry (MS) is a well-accepted means for analyzing glycans. Before glycan analysis by MS, several chemical derivatizations are generally carried out. These are classified into three categories; (1) labeling of the reducing end of glycans, (2) permethylation, and (3) sialic acid derivatization. Because sialic acid residues are unstable, they are easily lost during pretreatment and during or after ionization in a mass spectrometer. Sialic acid derivatization can prevent the loss of this residue. Recently, new types of sialic acid derivatization techniques have been developed, which allow straight-forward sialic acid linkage analysis (α2,3-/α2,6-linkages) as well as residue stabilization. This review summarizes the developments in sialic acid derivatization techniques, especially the varied methods of sialic acid linkage-specific derivatization.

Abstract LC–MS is one of the most important tools for the comprehensive characterization of N-glycans. Despite many efforts to speed up glycan analysis via optimized sample preparation (e.g., faster enzyme digestion in combination with instant or rapid labeling dyes), a major bottleneck remains the rather long measurement times of HILIC chromatography. Further complication arises from the necessity to concomitantly calibrate with an external standard to allow for accurate retention times and the conversion into more robust GU values. Here we demonstrate the use of an internal calibration strategy for HILIC chromatography to speed up glycan analysis. By reducing the number of utilized dextran oligosaccharides, the calibrant can be spiked directly into the sample such that external calibration runs are no longer required. The minimized dextran ladder shows accurate GU calibration with a minor deviation of well below 1% and can be applied without modifications in sample preparation or data processing. We further demonstrate the simultaneous use of the minimized dextran ladder as calibrant for the estimation of CCS values in traveling wave ion mobility spectrometry. In both cases, the minimized dextran ladder enables the measurement of calibrant and sample in a single HPLC run without losing information or accuracy.

Capillary zone electrophoresis (CZE) based on electrophoretic mobility in the liquid phase and ion mobility spectrometry (IMS) based on mobilities in the gas phase are both powerful techniques for the separation of complex samples. Protein glycosylation is one of the most common post-translational modifications associated with a wide range of biological functions and human diseases. Due to their high structural variability, the analysis of glycans still represents a challenging task. In this work, the first on-line coupling of CZE with drift tube ion mobility-mass spectrometry (DTIM-MS) has been perfomed to further improve separation capabilities for the analysis of native and 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled N-glycans. In this way, a complexity of glycan signals was revealed which could not be resolved by these techniques individually, shown for both native and APTS-labeled glycans. Each individual glycan signal separated in CZE exhibited an unexpectedly high number of peaks observed in the IMS dimension. This observation could potentially be explained by the presence of isomeric forms, including different linkages, and/or gas-phase conformers. In addition, the type of sialic acid attached to glycans has a significant impact on the obtained drift time profile. Furthermore, the application of α2-3 neuraminidase enabled the partial assignment of peaks in the arrival time distribution considering their sialic acid linkages (α2-3/α2-6). This work is a showcase for the high potential of CZE-DTIM-MS, which is expected to find various applications in the future.

Background Sulfate modification of N -glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N -glycan modification and its functions remain largely unexplored. Characterization of N -glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N -glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO 4 on N -glycans. Results Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO 4 when it is in a terminal position on an N -glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N -glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO 4 (but not asulfated GlcNAc) from a terminal position on N -glycans. Finally, the use of these enzymes to detect the presence of sulfated N -glycans by xCGE-LIF is demonstrated. Conclusion The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO 4 in N -glycan structural analyses.

Fabrazyme (agalsidase beta) is a therapeutic enzyme whose clinical efficacy is contingent upon its complex N-glycosylation patterns. Nevertheless, comprehensive glycosylation profiling remains challenging due to high site-specific heterogeneity. To address this, three orthogonal liquid chromatography–mass spectrometry (LC-MS) approaches were employed: (1) released N-glycan analysis with fluorescence detection and MS annotation, (2) site-specific glycopeptide mapping, and (3) intact protein MS. The released glycan profiling method was validated for reproducibility, intermediate precision, and inter-laboratory transferability, thereby enabling reliable separation and quantification of neutral, phosphorylated, and sialylated species. Glycopeptide mapping revealed distinct site-specific distributions: N108 was found to predominantly carry sialylated complex glycans; N161 was enriched in phosphorylated oligomannose structures; and N184 displayed the highest heterogeneity, including bisphosphorylated and sialylated glycans. Intact protein analysis was performed on both intact and desialylated Fabrazyme, thereby enabling confirmation of glycan assignments. Desialylation reduced spectral complexity, thereby facilitating accurate mass matching with a combinatorial library generated from glycopeptide-level data. The complementary use of these three analytical levels provides a comprehensive view of Fabrazyme glycosylation, offering a robust reference for quality control and biosimilar development.