N-Glycomic Analysis of the Cell Shows Specific Effects of Glycosyl Transferase Inhibitors.

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N-glycans of the glycocalyx. Glycomic analysis using liquid chromatography-mass spectrometry (LC-MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl-N-acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N-glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell-cell interactions.

Multivalent glycan arrays.

Glycan microarrays have become a powerful technology to study biological processes, such as cell-cell interaction, inflammation, and infections. Yet, several challenges, especially in multivalent display, remain. In this introductory lecture we discuss the state-of-the-art glycan microarray technology, with emphasis on novel approaches to access collections of pure glycans and their immobilization on surfaces. Future directions to mimic the natural glycan presentation on an array format, as well as in situ generation of combinatorial glycan collections, are discussed.

Development of a filter-aided extraction method coupled with glycosylamine labeling to simplify and enhance high performance liquid chromatography-based N-glycan analysis.

Efficient sample pretreatment of N-glycans from glycoproteins is essential but challenging due to the limitations of existing tedious and laborious methods in N-glycomics. This study aimed to establish a filter-aided extraction method coupled with glycosylamine AQC labeling for a simple and rapid direct HPLC-FLD-based analysis of N-glycans. The developed method was demonstrated to be simpler and more sensitive compared to previous HILIC SPE purification method coupled with glycosylamine labeling. It has been validated with wild-type N-glycans from human transferrin and RNase B and then was successfully applied to investigate N-glycan profiles of the transferrin in human serum and a monoclonal antibody (mAb). Results showed good applicability of the method for complex samples. Additionally, this method is compatible with the replicate determination of N-glycan samples to assess the high-throughput analysis of glycan variability in mAb sample.

GlyConnect: Glycoproteomics Goes Visual, Interactive, and Analytical.

Knowledge of glycoproteins, their site-specific glycosylation patterns, and the glycan structures that they present to their recognition partners in health and disease is gradually being built on using a range of experimental approaches. The data from these analyses are increasingly being standardized and presented in various sources, from supplemental tables in publications to localized servers in investigator laboratories. Bioinformatics tools are now needed to collect these data and enable the user to search, display, and connect glycomics and glycoproteomics to other sources of related proteomics, genomics, and interactomics information. We here introduce GlyConnect ( https://glyconnect.expasy.org/ ), the central platform of the Glycomics@ExPASy portal for glycoinformatics. GlyConnect has been developed to gather, monitor, integrate, and visualize data in a user-friendly way to facilitate the interpretation of collected glycoscience data. GlyConnect is designed to accommodate and integrate multiple data types as they are increasingly produced.

Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses.

Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycans are produced through a complicated nontemplate driven process involving the competition of enzymes that extend the nascent chain. The large diversity of structures, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies of glycans all conspire to make the analysis arguably much more difficult than any other biopolymer. Furthermore, the large number of glycoforms associated with a specific protein site makes it more difficult to characterize than any post-translational modification. Nonetheless, there have been significant progress, and advanced separation and mass spectrometry methods have been at its center and the main reason for the progress. While glycomic and glycoproteomic analyses are still typically available only through highly specialized laboratories, new software and workflow is making it more accessible. This review focuses on the role of mass spectrometry and separation methods in advancing glycomic and glycoproteomic analyses. It describes the current state of the field and progress toward making it more available to the larger scientific community.

Focused Glycomic Profiling With an Integrated Microfluidic Lectin Barcode System.

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges associated with the analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and the speed of lectin microarrays. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, the CA125 protein purified from ovarian cancer cell lines, and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated "sample-to-answer" microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glycobiomarker development.

Glycosaminoglycanomics: where we are.

Glycosaminoglycans regulate numerous physiopathological processes such as development, angiogenesis, innate immunity, cancer and neurodegenerative diseases. Cell surface GAGs are involved in cell-cell and cell-matrix interactions, cell adhesion and signaling, and host-pathogen interactions. GAGs contribute to the assembly of the extracellular matrix and heparan sulfate chains are able to sequester growth factors in the ECM. Their biological activities are regulated by their interactions with proteins. The structural heterogeneity of GAGs, mostly due to chemical modifications occurring during and after their synthesis, makes the development of analytical techniques for their profiling in cells, tissues, and biological fluids, and of computational tools for mining GAG-protein interaction data very challenging. We give here an overview of the experimental approaches used in glycosaminoglycomics, of the major GAG-protein interactomes characterized so far, and of the computational tools and databases available to analyze and store GAG structures and interactions.

High-Throughput Analysis of the Plasma N-Glycome by UHPLC.

The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation.

Algorithms and design strategies towards automated glycoproteomics analysis.

Glycoproteomics involves the study of glycosylation events on protein sequences ranging from purified proteins to whole proteome scales. Understanding these complex post-translational modification (PTM) events requires elucidation of the glycan moieties (monosaccharide sequences and glycosidic linkages between residues), protein sequences, as well as site-specific attachment of glycan moieties onto protein sequences, in a spatial and temporal manner in a variety of biological contexts. Compared with proteomics, bioinformatics for glycoproteomics is immature and many researchers still rely on tedious manual interpretation of glycoproteomics data. As sample preparation protocols and analysis techniques have matured, the number of publications on glycoproteomics and bioinformatics has increased substantially; however, the lack of consensus on tool development and code reuse limits the dissemination of bioinformatics tools because it requires significant effort to migrate a computational tool tailored for one method design to alternative methods. This review discusses algorithms and methods in glycoproteomics, and refers to the general proteomics field for potential solutions. It also introduces general strategies for tool integration and pipeline construction in order to better serve the glycoproteomics community. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:475-498, 2017.

Changes in cellular glycosylation of leukemia cells upon treatment with acridone derivatives yield insight into drug action.

A new acridone derivative 2-aminoacetamido-10-(3, 5-dimethoxy)-benzyl-9(10H)-acridone hydrochloride (8a) has been shown to have potent antitumor activity. In order to understand the underlying action mechanism of 8a, three compounds of the same class with structures optimized step-by-step, 9(10H)-acridone (A), 10-(3,5-dimethoxy) benzyl-9(10H)-acridone (I) and 8a, were exposed to CCRF-CEM leukemia cell to determine the N-glycosylation changes using the microfluidic HPLC-chip-TOF MS platform. N-Glycans from whole cell lysates (WCL) and cell membranes (CM) were analyzed using isomer-sensitive chip-based porous graphitized carbon nano-LC/MS. A total of 223 N-glycan compositions and 398 N-glycan compounds were identified. Comparison of the two analyses showed that more apparent changes were observed in the CM compared with WCL, suggesting that CM may be a more sensitive indicator of changes in glycosylation. Upon 8a exposure to CCRF-CEM cells, a significant decrease in high-mannose-type glycans was observed. Different expressions of oligosaccharyltransferase subunits appear to play a key functional role in regulating the hypoglycosylation and contribute to the action mechanism of 8a. Taken together our findings suggest that glycosylation is strongly affected by therapeutic potency and can be used as possible biomarkers for monitoring toxicity and antitumor activity of 8a.

Protecting group-free immobilization of glycans for affinity chromatography using glycosylsulfonohydrazide donors.

A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are known, they may provide certain advantages or disadvantages in how the sugar is attached to the affinity matrix. Additionally, the products of some methods may be hard to characterize chemically due to non-specific reactions. The lack of specificity in standard immobilization reactions makes affinity chromatography with expensive oligosaccharides challenging. As a result, methods for specific and efficient immobilization of oligosaccharides remain of interest. Herein, we present a method for the immobilization of saccharides using N'-glycosylsulfonohydrazide (GSH) carbohydrate donors. We have compared GSH immobilization to known strategies, including the use of divinyl sulfone (DVS) and cyanuric chloride (CC), for the generation of affinity matrices. We compared immobilization methods by determining their immobilization efficiency, based on a comparison of the mass of immobilized carbohydrate and the concentration of active binding sites (determined using lectins). Our results indicate that immobilization using GSH donors can provide comparable amounts of carbohydrate epitopes on solid support while consuming almost half of the material required for DVS immobilization. The lectin binding capacity observed for these two methods suggests that GSH immobilization is more efficient. We propose that this method of oligosaccharide immobilization will be an important tool for glycobiologists working with precious glycan samples purified from biological sources.

Automated, high-throughput serum glycoprofiling platform.

Complex carbohydrates are rapidly becoming excellent biomarker candidates because of their high sensitivity to pathological changes. However, the discovery of clinical glycobiomarkers has been slow, due to the scarcity of high-throughput glycoanalytical workflows that allow rapid glycoprofiling of large clinical sample sets. To generate high-quality quantitative glycomics data in a high-throughput fashion, we have developed a robotized platform for rapid serum-based N-glycan sample preparation. The sample preparation workflow features a fully automated, rapid glycoprotein denaturation followed by sequential enzymatic glycan release, glycan purification on solid-supported hydrazide and fluorescent labelling. This allows accurate glycan quantitation by ultra-high performance liquid chromatography (UPLC). The sample preparation workflow was automated using an eight-channel Hamilton Robotics liquid handling workstation, allowing the preparation of almost 100 samples in 14 hours with excellent reproducibility and thus should greatly facilitate serum-based glyco-biomarker discovery.

Development and Applications of the Lectin Microarray.

The lectin microarray is an emerging technology for glycomics. It has already found maximum use in diverse fields of glycobiology by providing simple procedures for differential glycan profiling in a rapid and high-throughput manner. Since its first appearance in the literature in 2005, many application methods have been developed essentially on the same platform, comprising a series of glycan-binding proteins immobilized on an appropriate substrate such as a glass slide. Because the lectin microarray strategy does not require prior liberation of glycans from the core protein in glycoprotein analysis, it should encourage researchers not familiar with glycotechnology to use glycan analysis in future work. This feasibility should provide a broader range of experimental scientists with good opportunities to investigate novel aspects of glycoscience. Applications of the technology include not only basic sciences but also the growing fields of bio-industry. This chapter describes first the essence of glycan profiling and the basic fabrication of the lectin microarray for this purpose. In the latter part the focus is on diverse applications to both structural and functional glycomics, with emphasis on the wide applicability now available with this new technology. Finally, the importance of developing advanced lectin engineering is discussed.

Cyto-sensing in electrochemical lab-on-paper cyto-device for in-situ evaluation of multi-glycan expressions on cancer cells.

A novel electrochemical lab-on-paper cyto-device (ELPCD) was fabricated to demonstrate sensitive and specific cancer cell detection as well as in-situ monitoring of multi-glycans on living cancer cells. In this ELPCD, aptamers modified three-dimensional macroporous Au-paper electrode (Au-PE) was employed as the working electrode for specific and efficient cancer cell capture. Using a sandwich format, sensitive and reproducible cell detection was achieved in this ELPCD on the basis of the electrochemical signal amplification of the Au-PE and the horseradish peroxidase-lectin electrochemical probe. The ELPCD displayed excellent analytical performance for the detection of four K562 cells with a wide linear calibration range from 550 to 2.0×10(7) cells mL(-1). Then, this ELPCD was successfully applied to determine cell-surface multi-glycans in parallel and in-situ monitor multi-glycans expression on living cells in response to drug treatment through in-electrode 3D cell culture. The proposed method provides promising application in decipherment of the glycomic codes as well as clinical diagnosis and treatment in early process of cancer.

Phosphoneurofilament heavy chain and N-glycomics from the cerebrospinal fluid in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified. METHODS: We have quantified by ELISA the biomarker phosphoneurofilament heavy chain (pNFH) in the cerebrospinal fluid (CSF) of ALS patients (n=29) and age-matched control patients with other diseases (n=19) by ELISA. Furthermore, we compared protein N-glycosylation of the CSF in ALS patients and controls, by applying a glycomics approach based on liquid chromatography and mass spectrometry. RESULTS: pNFH levels were significantly higher in ALS patients in comparison with controls (P<0.0001) in particular in fast progressors. The N-glycans found in the CSF were predominantly complex diantennary with sialic acid in α2,3- and α2,6-linkage, and bisecting N-acetylglucosamine-containing structures as well as peripherally fucosylated structures were found. As compared with controls the ALS group had a significant increase of a peak composed of the monosialylated diantennary glycans A2G2S(6)1 and FA2G2S(3)1 (P=0.0348). CONCLUSIONS: Our results underscore the value of pNFH as a biomarker in ALS. In addition, we identified a variation of the N-glycosylation pattern in ALS, suggesting that this change should be explored in future studies as potential biomarker.

Frontal affinity chromatography: a unique research tool for biospecific interaction that promotes glycobiology.

Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described.

New fabrication and applications of carbohydrate arrays.

Carbohydrate arrays are used as high-throughput screening platforms to study the carbohydrate-mediated recognition events for glycobiology. The polysaccharide arrays are easy to fabricate by non-covalently or covalently immobilizing polysaccharides onto array surfaces because polysaccharides have hydrophobic interactions. Oligosaccharides must be derived and covalently or non-covalently immobilized onto array surfaces to fabricate oligosaccharide arrays because they have hydrophilic interactions. At the moment, carbohydrate arrays are mainly used to study the carbohydrate-protein interactions and carbohydrate-binding lectins or antibodies, which are possible to be applied to clinics and diagnoses. This review mainly summed up the new fabrication strategies of carbohydrate arrays and their applications in recent four years.

Novel nanomaterials used for sample preparation for protein analysis.

Sample preparation is of vital importance for proteomic analysis because of the high complexity of biological samples. The rapid development of novel nanomaterials with various compositions, morphologies, and proper surface modifications provides a category of powerful tools for the sample preparation for protein analysis. In this paper, we have summarized recent progresses for the applications of novel nanomaterials in sample preparation for the analysis of proteomes, especially for phosphoproteomes, glycoproteomes, and peptidoms. Several kinds of novel nanomaterials were also discussed for their use in other kinds of proteomics analysis.