Ion Mobility Mass Spectrometry-Based Disaccharide Analysis of Glycosaminoglycans.

Glycosaminoglycans (GAGs) are linear and acidic polysaccharides. They are ubiquitous molecules, which are involved in a wide range of biological processes. Despite being structurally simple at first glance, with a repeating backbone of alternating hexuronic acid and hexosamine dimers, GAGs display a highly complex structure, which predominantly results from their heterogeneous sulfation patterns. The commonly applied method for compositional analysis of all GAGs is "disaccharide analysis." In this process, GAGs are enzymatically depolymerized into disaccharides, derivatized with a fluorescent label, and then analysed through liquid chromatography. The limiting factor in the high throughput analysis of GAG disaccharides is the time-consuming liquid chromatography. To address this limitation, we here utilized trapped ion mobility-mass spectrometry (TIM-MS) for the separation of isomeric GAG disaccharides, which reduces the measurement time from hours to a few minutes. A full set of disaccharides comprises twelve structures, with eight possessing isomers. Most disaccharides cannot be differentiated by TIM-MS in underivatized form. Therefore, we developed chemical modifications to reduce sample complexity and enhance differentiability. Quantification is performed using stable isotope labelled standards, which are easily available due to the nature of the performed modifications.

Variability in the composition of porcine mucosal heparan sulfates.

Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.

Preparation and Characterization of Heparan Sulfate-Derived Oligosaccharides to Investigate Protein-GAG Interaction and HS Biosynthesis Enzyme Activity.

Heparan sulfate chains are complex and structurally diverse polysaccharides that interact with a large number of proteins, thereby regulating a vast array of biological functions. Understanding this activity requires obtaining oligosaccharides of defined structures. Here we describe methods for isolating, engineering, and characterizing heparan sulfate-derived oligosaccharides and approaches based on high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and bio-layer interferometry (BLI) to study their structures, modifications, and interactions.

Beware, commercial chondroitinases vary in activity and substrate specificity.

Chondroitin sulfate (CS)and dermatan sulfate (DS) are negatively charged polysaccharides found abundantly in animal tissue and have been extensively described to play key roles in health and disease. The most common method to analyze their structure is by digestion into disaccharides with bacterial chondroitinases, followed by chromatography and/or mass spectrometry. While studying the structure of oncofetal CS, we noted a large variation in the activity and specificity of commercially available chondroitinases. Here studied the kinetics of the enzymes and used high-performance liquid chromatography-mass spectrometry to determine the di- and oligosaccharide products resulting from the digestion of commercially available bovine CS A, shark CS C and porcine DS, focusing on chondroitinases ABC, AC and B from different vendors. Application of a standardized assay setup demonstrated large variations in the enzyme-specific activity compared to the values provided by vendors, large variation in enzyme specific activity of similar enzymes from different vendors and differences in the extent of cleavage of the substrates and the generated products. The high variability of different chondroitinases highlights the importance of testing enzyme activity and monitoring product formation in assessing the content and composition of chondroitin and DSs in cells and tissues.

Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow-/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia.

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O2) and hypoxic (5% O2) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

Glycosaminoglycanomic profiling of human milk in different stages of lactation by liquid chromatography-tandem mass spectrometry.

Glycans in human milk serve several important biological functions that promote infant health. As kind of important glycans, glycosaminoglycans (GAGs) are a complex family of polyanionic carbohydrate, participating in a variety of critical physiological and pathological processes. In this study, the content and the detailed composition of human milk GAGs from Chinese mothers in different stages of lactation, based on a liquid chromatography-tandem mass spectrometry approach was investigated. The results showed that the GAG fraction in the human milk samples was very complex as it was composed of heparan sulfate, chondroitin sulfate, and hyaluronic acid. With lactation extending, the total amount of GAGs in human milk decreased. This study provided an important guide for the demands of GAGs during different stages of lactation. The results were also beneficial for studies on the composition and functional properties of infant formula.

Overview of the quality control of chondroitin sulfate / 中国药学杂志

Chondroitin sulfate (CS) is an anionic linear polysaccharide having natural bioactivity, which is widely utilized as a drug and nutraceutical. Due to the structure complexity and source variety of chondroitin sulfate, the specifications of chondroitin sulfate in different pharmacopoeias were different. The related references published in recent years and pharmacopeias were analyzed, sorted, and summarized. This review summarized the methods and limits that were used to for the assay and inspection of impurities, disaccharide and relative molecular weight of chondroitin sulfate. This article would be beneficial to improve the quality control system of chondroitin sulfate.

Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.

Heparinase Digestion-based Disaccharide Analysis of Clinical Heparin and Heparinoids Drug / 分析化学

Heparin and low molecular weight heparin have been widely used in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS(6S)-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are drugs prepared from heparin active pharmaceutical ingredient ( API ) that is prepared from crude heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller sizes. As a result, low molecular weight heparins are sharing the same major disaccharides but have different reducing and non-reducing ends. In current study, we focused on the disaccharide compositional analysis of clinically used heparin and heparin-based drugs. HeparinaseⅠ,II, and Ⅲ were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, Fondaparinux sodium into disaccharides. All the degraded products were analyzed by strong anion high perforance liquid chromatography ( SAX-HPLC) coupled with an UV-detector. Commercially available unsaturated disaccharide standards were then used for structral identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin, and Fondaparinux were confirmed by reversed-phase ion pair HPLC coupled with mass spectrometry. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.

Cell-Based Microscale Isolation of Glycoaminoglycans for Glycomics Study.

Glycomics research requires the isolation of glycans from cells for structural characterization and functional studies of the glycans. A method for cell-based microscale isolation and quantification of highly sulfated, moderately sulfated, and nonsulfated glycosaminoglycans (GAGs) was developed using Chinese hamster ovary (CHO) cells. This microscale isolation relies on a mini-strong anion exchange spin column eluted stepwise with different concentrations of sodium chloride solution. Hyaluronic acid, chondroitin sulfate, and heparin were used to optimize the isolation of the endogenous glycosaminoglycans in CHO cells. This method can also be used to determine the presence of nonsulfated GAGs including heparosan, hyaluronic acid, and nonsulfated chondroitin.