Glucose Unit Values Research Articles

Labeling of Mucin-Type O-Glycans for Quantification Using Liquid Chromatography and Fluorescence Detection.

O-glycosylation is a common post-translational modification that is essential for the defensive properties of mucus barriers. Incomplete and altered O-glycosylation is often linked to severe diseases, such as cancer, cystic fibrosis, and chronic obstructive pulmonary disease. Originating from a nontemplate-driven biosynthesis, mucin-type O-glycan structures are very complex. They are often present as heterogeneous mixtures containing multiple isomers. Therefore, the analysis of complex O-glycan mixtures usually requires hyphenation of orthogonal techniques such as liquid chromatography (LC), ion mobility spectrometry, and mass spectrometry (MS). However, MS-based techniques are mainly qualitative. Moreover, LC separation of O-glycans often lacks reproducibility and requires sophisticated data treatment and analysis. Here we present a mucin-type O-glycomics analysis workflow that utilizes hydrophilic interaction liquid chromatography for separation and fluorescence labeling for detection and quantification. In combination with mass spectrometry, a detailed analysis on the relative abundance of specific mucin-type O-glycan compositions and features, such as fucose, sialic acids, and sulfates, is performed. Furthermore, the average number of monosaccharide units of O-glycans in different samples was determined. To demonstrate universal applicability, the method was tested on mucins from different tissue types and mammals, such as bovine submaxillary mucins, porcine gastric mucins, and human milk mucins. To account for day-to-day retention time shifts in O-glycan separations and increase the comparability between different instruments and laboratories, we included fluorescently labeled dextran ladders in our workflow. In addition, we set up a library of glucose unit values for all identified O-glycans, which can be used to simplify the identification process of glycans in future analyses.

Changes of IgG N-Glycosylation in Thyroid Autoimmunity: The Modulatory Effect of Methimazole in Graves' Disease and the Association With the Severity of Inflammation in Hashimoto's Thyroiditis.

The N-glycome of immunoglobulin G (IgG), the most abundant glycoprotein in human blood serum, reflects pathological conditions of autoimmunity and is sensitive to medicines applied in disease therapy. Due to the high sensitivity of N-glycosylation, the IgG N-glycan profile may serve as an indicator of an ongoing inflammatory process. The IgG structure and its effector functions are strongly dependent on the composition of N-glycans attached to the Fc fragment, and the binding of antigens is regulated by Fab sugar moieties. Because of the crucial role of N-glycans in IgG function, remodeling of its N-oligosaccharides can induce pathological changes that ultimately contribute to the development of autoimmunity; restoration of their physiological structure is critical to the reduction of disease symptoms. Our recently published data have shown that the pathology of autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), is accompanied by alterations of the composition of IgG N-glycans. The present study is a more in-depth investigation of IgG glycosylation in both AITDs, designed to determine the relationship between the severity of thyroid inflammation and IgG N-glycan structures in HT, and to assess the impact of immunosuppressive therapy on the N-glycan profile in GD patients. The study material consisted of human serum samples collected from donors with elevated anti-thyroglobulin (Tg) and/or anti-thyroperoxidase (TPO) IgGs without symptoms of hypothyroidism (n=68), HT patients characterized by high autoantibody titers and advanced destruction of the thyroid gland (n=113), GD patients with up-regulated IgG against thyroid-stimulating hormone receptor (TSHR) before (n=62) and after (n=47) stabilization of TSH level as a result of methimazole therapy (study groups), and healthy donors (control group, n=90). IgG was isolated from blood serum using protein G affinity chromatography. N-glycans were released from IgG by PNGase F digestion and analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) after 2-aminobenzamide (2-AB) labeling. UPLC-MS chromatograms were integrated into 25 peaks (GP) in the Waters UNIFI Scientific Information System, and N-glycans were assigned based on the glucose unit values and mass-to-charge ratios (m/z) of the detected ions. The Kruskal-Wallis non-parametric test was used to determine the statistical significance of the results (p<0.05). The obtained results suggest that modifications of IgG sialylation, galactosylation and core-fucosylation are associated with the severity of HT symptoms. Methimazole therapy implemented in GD patients affected the IgG N-glycan profile; as a result, the content of the sialylated and galactosylated oligosaccharides with core fucose differed after treatment. Our results suggest that N-glycosylation of IgG undergoes dynamic changes during the intensification of thyroiditis in HT, and that in GD autoimmunity it is affected significantly by immunosuppressive therapy.

Expanding the capillary electrophoresis-based glucose unit database of the GUcal app.

GUcal is a standalone application for automatically calculating the glucose unit (GU) values for separated N-glycan components of interest in an electropherogram and suggests their tentative structures by utilizing an internal database. We have expanded the original database of GUcal by integrating all publicly available capillary electrophoresis (CE) data in the GlycoStore collection (https://www.glycostore.org) and with in-house measured GU values. The GUcal app is freely available online (https://www.gucal.hu) and readily facilitates CE-based high throughput GU value determination for first line structural elucidation.

Combining Glucose Units, m/z, and Collision Cross Section Values: Multiattribute Data for Increased Accuracy in Automated Glycosphingolipid Glycan Identifications and Its Application in Triple Negative Breast Cancer.

Glycan head-groups attached to glycosphingolipids (GSLs) found in the cell membrane bilayer can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. To identify such markers, comprehensive analyses of glycan structures must be undertaken. Conventional analyses of fluorescently labeled glycans using hydrophilic interaction high-performance liquid chromatography (HILIC) coupled with mass spectrometry (MS) provides relative quantitation and has the ability to perform automated glycan assignments using glucose unit (GU) and mass matching. The use of ion mobility (IM) as an additional level of separation can aid the characterization of closely related or isomeric structures through the generation of glycan collision cross section (CCS) identifiers. Here, we present a workflow for the analysis of procainamide-labeled GSL glycans using HILIC-IM-MS and a new, automated glycan identification strategy whereby multiple glycan attributes are combined to increase accuracy in automated structural assignments. For glycan matching and identification, an experimental reference database of GSL glycans containing GU, mass, and CCS values for each glycan was created. To assess the accuracy of glycan assignments, a distance-based confidence metric was used. The assignment accuracy was significantly better compared to conventional HILIC-MS approaches (using mass and GU only). This workflow was applied to the study of two Triple Negative Breast Cancer (TNBC) cell lines and revealed potential GSL glycosylation signatures characteristic of different TNBC subtypes.

Structural identification of N-linked carbohydrates using the GUcal application: A tutorial

In recent years, analytical glycomics gained a significant role due to the rapidly increasing number of glycoproteins introduced as biopharmaceuticals. One of the frequently used methods for the analysis of complex carbohydrates is capillary electrophoresis with laser induced fluorescent detection (CE-LIF). CE-LIF is a high resolution separation technique with excellent sensitivity capable of discriminating between closely related positional and linkage carbohydrate isomers. Individual glycan structures corresponding to the separated peaks in an electropherogram are identified according to their glucose unit (GU) values by mining the built in database. This tutorial introduces the practical use of the GUcal application, a recently developed glycoinformatics tool, which automatically calculates GU values for all sample components of interest in an electropherogram using either the bracketing standard approach or the recently published triple internal standard method. Furthermore, a worked example demonstrates the way glycan structural elucidation of human immunoglobulin G is processed with the help of this simple and rapid GU value calculation application. SignificanceBiopharmaceuticals have seen something of tremendous development in recent years, which governs the parallel blooming of analytical glycomics. One of the frequently used methods for the analysis of complex carbohydrates is capillary electrophoresis with laser induced fluorescent detection (CE-LIF). CE-LIF is a high resolution separation technique with excellent sensitivity capable of discriminating between closely related positional and linkage carbohydrate isomers. While CE instrumentation is well developed, the related bioinformatics tools are lagging behind. According to our best knowledge, this is the first tutorial paper on the recently disseminated GUcal application, which automatically calculates GU values for all sample components of interest in an electropherogram using either the bracketing standard approach or the new triple internal standard method. On the top the step-by-step instruction how to use the application, the paper includes a worked example demonstrates the way glycan structure elucidation of human immunoglobulin G is processed with the help of the simple and rapid GU value calculation of this new application. After very short training period, the software can be used readily for cutting-edge glycomics of exploratory research or high-throughput routine analysis.

Triple-Internal Standard Based Glycan Structural Assignment Method for Capillary Electrophoresis Analysis of Carbohydrates.

Despite the ever growing use of capillary electrophoresis in biomedical research and the biopharmaceutical industry, the development of data interpretation methods is lagging behind. In this paper we report the design and implementation of a coinjected triple-internal standard method to alleviate the need of an accompanying run of the maltooligosaccharide ladder for glucose unit (GU) calculation. Based on the migration times of the coinjected standards of maltose, maltotriose, and maltopentadecaose (bracketing the peaks of interest), a data processing approach was designed and developed to set up a virtual ladder that was used for GU calculation. The data processing was tested in terms of the calculated GU values of human IgG glycans, and the resulting relative standard deviation was ≤1.07%. This approach readily supports high-throughput capillary electrophoresis systems by significantly speeding up the processing time for glycan structural assignment.

Molecular and cellular mechanisms of profile changes of charged blood plasma free oligosaccharides in myeloproliferative disorders

Free oligosaccharides (FOS) are unbound to proteins or lipids structural analogs of their glycans. FOS appear as by-products of endoplasmic reticulum synthesis, cell control folding with endoplastic reticulum-associated degradation and lysosomal/endosomal breakdown of glycoconjugates. They may be either neutral or negatively charged depending on the way of their formation. Charged FOS appear during degradation of glycoconjugates in the lysosomal/endosomal system and are natural substrates for lysosomal sialidase-1. FOS are formed inside the cell but some of them can get into the extracellular space, and then into the blood and urine, where both neutral and charged structures were found. Secretion of charged FOS outside of the cell is most likely to be caused by the lysosomal exocytosis. The activity of neuraminidase-1 is known to be connected with the intensity of the lysosomal exocytosis. In our previous studies, it was found that HPLC-profiles of charged FOS were specifically changed in acute and chronic myeloproliferative blood disorders. The objective of this work was to analyze the molecular and cellular mechanisms of these changes and to test the hypothesis of their association with the activity change of lysosomal neuraminidase-1. Plasma samples of patients with acute and chronic leukaemia – polycytaemia vera, chronic idiopathic myelofibrosis, hypoplastic anaemia, myelodysplastic syndrome with transformation, acute lymphatic and acute myelomonocytic leukaemia – were collected for investigation. Plasma samples of practically healthy volunteers were obtained and used for comparison. After plasma deproteinization and FOS purification the oligosaccharides were labelled with anthranilic acid (2-AA), separated into the neutral and charged fractions with QAE Sephadex (Q25-120) chromatography and analysed using high-performance liquid chromatography (HPLC). Glucose unit values were determined following comparison with a 2-AA-labelled glucose oligomer ladder derived from a partial hydrolysate of dextran as an external standard. The data were collected and processed using Empower software. The charged FOS were digested with the sialidase from Arthrobacter ureafaciens. 2-AA – labelled free oligosaccharides from transferrin were used as an external standard for the structure decoding. In this paper, the changes in neraminidase-1 activity was first demonstrated on natural substrates, free oligosaccharides, in vivo. Chromatographic profiles of charged plasma FOS of patients with acute and chronic hematological malignancies revealed decreasing of neuraminidase-1 activity and increasing of the lysosomal exocytosis. Thus, chromatographic profiles of charged plasma FOS appeared to be a sensitive parameter of the lysosomal/endosomal status in normal or pathological states and to open up new prospects for their applications to the development of anti-cancer drugs designed to block the work of the lysosomal/endosomal system and monitoring of their action.

Chromatographic profiles of blood plasma free oligosaccharides in patients with cardiovascular disease

Free oligosaccharides (FOS) are unbound structural analogs of glycans of glycoconjugates. There are several sources of them inside the cell: 1) multistep pathways of N-glycosylation, 2) the cell quality control and endoplastic reticulum-associated degradation of mis-glycosylated and/or misfolded glycoproteins, 3) lysosomal degradation of mature glycoconjugates. Some of these FOS are the earliest indicators of potential glycosylation alterations that would be revealed in the course of the cell quality control and the endoplastic reticulum-associated degradation. Ischemia and hypertension cause stress of intracellular organelles leading to disruption of their functions. The main objective of the work was the characterization of free oligosaccharides (FOS) in plasma obtained from patients with cardiovascular diseases compared to those from healthy subjects to evaluate the potential of these compounds for diagnostics. Chromatographic profiles of FOS composed of 4–12 monosaccharides were obtained and analyzed for quantitative and qualitative differences between the samples. After plasma deproteinization and FOS purification the oligosaccharides were labelled with anthranilic acid (2-AA), separated into the neutral and charged with QAE Sephadex (Q25-120) chromatography and analysed using high-performance liquid chromatography (HPLC). Glucose unit values were determined following comparison with a 2-AA-labelled glucose oligomer ladder derived from a partial hydrolysate of dextran as an external standard. The data were collected and processed using Empower software. The charged FOS were digested with the sialidase from Arthrobacter ureafaciens. 2-AA – labelled free oligosaccharides from transferrin were used as an external standard for the structure decoding. The profiles obtained were compared with intracellular free oligosaccharides of known structures and with the glycan structures and their descriptions in the databases GlycoBase and EUROCarbDB. These approaches allowed predicting a range of glycan structures for each of the main peaks of HPLC profiles of plasma free oligosaccharides and managing ways for their future experimental analysis. In the case of cardiovascular disorders, HPLC profiles of FOS revealed a changing pattern of heterogeneity, depending on the severity of the disease. Three main enlarged glycan species in the netral fraction and one peak in the charged fraction distinguished the FOS of the patients from those of the healthy volunteers. It has been revealed that the neutral marker peaks were represented by polimannose glycans with 5–7 mannose residues and 1–2 residues of N-acetylglucosamine, and one of the major peaks of the charged fraction – by two-antennary complex N-glycan with two sialic acid residues. The study of free oligosaccharides of blood plasma is a new field of glycobiology allowing an evaluation of an organism state at the level of the cell organelle functional status and openning up broad prospects for finding early diagnostic and prognostic markers of cardiac insufficiency.

Hydrophilic interaction liquid chromatography of anthranilic acid-labelled oligosaccharides with a 4-aminobenzoic acid ethyl ester-labelled dextran hydrolysate internal standard

Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant.

Method for milk oligosaccharide profiling by 2-aminobenzamide labeling and hydrophilic interaction chromatography

Although the properties of milk oligosaccharides have been of scientific interest for many years, their structural diversity presents a challenging analytical task. In the quest for a simple and robust technology to characterize the different oligosaccharides present in milk, we developed an analytical scheme based on their fluorescent labeling, pre-fractionation by weak anionic exchange chromatography and separation by hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC). HILIC relies on the hydrophilic potential of the molecule, which accounts for differences in properties such as molecular volume, lipophilic surface area, charge, composition, structure, linkage and oligosaccharide branching. The robustness of the methodology has been demonstrated using bovine colostrum oligosaccharides as a case study. Structural assignments for 37 free glycans, including 20 sialylated species, were obtained by a combination of HILIC-HPLC, exoglycosidase digestion and offline negative-ion mode mass spectrometry (MS)/MS. Parameters obtained for each glycan, including linkages, enzymatic digestion products and glucose unit values, will be added to GlycoBase, a public access database (http://glycobase.nibrt.ie/glycobase.html). This approach provides a basis for the analysis of free milk oligosaccharides in a fast and sensitive manner and could be adapted for an automated technology platform amenable to diverse environments. Indeed, our approach, in conjunction with bacterial-binding assays, can provide a better understanding of the structural elements required for biological activity of free milk oligosaccharides and could serve as a scientific basis for the selection of such bioactives from various food sources.

Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

CE is a high-resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37 degrees C, using > or =100 fold excess of fluorophore reagent, 8-aminopyrene-1,3,6-trisulfonic-acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:> or =100 ratio) maintained the >95% derivatization yield at 55 degrees C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55 degrees C during the derivatization process, and indicating that the kinetics of labeling at 55 degrees C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8-aminopyrene-1,3,6-trisulfonic-acid simplified the removal of excess reagent, important in both CE-LIF (electrokinetic injection bias) and CE-MS (ion suppression). Coupling CE- ESI-MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.

GlycoExtractor: A Web-Based Interface for High Throughput Processing of HPLC-Glycan Data

Recently, an automated high-throughput HPLC platform has been developed that can be used to fully sequence and quantify low concentrations of N-linked sugars released from glycoproteins, supported by an experimental database (GlycoBase) and analytical tools (autoGU). However, commercial packages that support the operation of HPLC instruments and data storage lack platforms for the extraction of large volumes of data. The lack of resources and agreed formats in glycomics is now a major limiting factor that restricts the development of bioinformatic tools and automated workflows for high-throughput HPLC data analysis. GlycoExtractor is a web-based tool that interfaces with a commercial HPLC database/software solution to facilitate the extraction of large volumes of processed glycan profile data (peak number, peak areas, and glucose unit values). The tool allows the user to export a series of sample sets to a set of file formats (XML, JSON, and CSV) rather than a collection of disconnected files. This approach not only reduces the amount of manual refinement required to export data into a suitable format for data analysis but also opens the field to new approaches for high-throughput data interpretation and storage, including biomarker discovery and validation and monitoring of online bioprocessing conditions for next generation biotherapeutics.

Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

A Rapid High-Resolution High-Performance Liquid Chromatographic Method for Separating Glycan Mixtures and Analyzing Oligosaccharide Profiles

A sensitive and reproducible HPLC technology has been developed, capable of resolving sub-picomolar quantities of mixtures of fluorescently labeled neutral and acidic glycans simultaneously and in their correct molar proportions. The elution positions of standard glycans were determined in glucose units with reference to a dextran ladder, and incremental values for the addition of monosaccharides to oligosaccharide cores were calculated. This information was used to interpret the full oligosaccharide profiles of glycoproteins in a predictive manner based on arm specificity, linkage, and monosaccharide composition. The technique was applied to several systems. For example, a family of glycans isolated from the human parotid gland was extensively resolved on the basis of type and extent of outer arm fucosylation. Second, a serum IgG glycan pool was resolved into 20 peaks which were analyzed simultaneously by sequentially digesting the pool of sugars with exoglycosidase enzymes. In addition, alterations in the glycosylation of IgG associated with rheumatoid arthritis were directly monitored. The reproducibility of the separation system, the predictability of glucose unit values, and the quantitative response of the detection system for individual fluorescently labeled glycans also allowed the automatic analysis of neutral sugars using combinations of enzymes as in the reagent array analysis method (RAAM). In addition, the simultaneous resolution of both acidic (sialylated) and neutral products from the RAAM digestion allowed direct analysis of sialylated glycans, eliminating the previous need to remove sialic acid residues in a preliminary step. Overall, the technologies described here represent a significant advance toward faster, more automated, and more detailed glycan analysis.