⿢The oxidized cellulose with different degree of oxidation (DO) were prepared.⿢Their DOs were determined and compared using IR and NMR.⿢The impact of DO on their structural properties were investigated in detail.Microcrystalline cellulose, a major component of cell wall of plants, is one of the most abundant natural materials, but the poor solubility of cellulose limits its applications. Cellulose is a linear glucan with exclusive β 1 ⿿ 4 linkage. Oxidation carried out with TEMPO⿿NaBr⿿NaClO system can selectively oxidize the C6 of glucose residues in cellulose. This modification improves polysaccharide solubility and other physicochemical properties. In this work, the impact of degree of oxidation on solubility, degree of crystallization, thermostability, molecular weight and the structures of the resulting oligosaccharide products of selectively oxidized cellulose were investigated using x-ray diffraction, thermogravimetric analysis, gel permeation chromatography⿿multiple angle laser light scattering and ultrahigh performance liquid chromatography⿿electrospray⿿quadrupole/time of flight⿿mass spectrometry, respectively. The physicochemical properties of selectively oxidized cellulose having different degrees of oxidation were carefully characterized providing a theoretical foundation for the more accurate selection of applications of oxidized celluloses.

•The MW of heparin and LMWHs determined by SEC-MALS is not accurate.•The ion-paired cations on heparin and LMWHs were not considered previously.•A comprehensive strategy with SEC-MALS/RI and SEC-ICP/MS was developed.•Polysaccharide/cations of heparin were measured with two methods, respectively.•MW/mass recovery of heparin/LMWHs were determined accurately with this strategy.Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. Low molecular weight heparins (LMWHs), heparins partially depolymerized using different processes, are widely used as clinical anticoagulants. Qualitative molecular weight (MW) and quantitative mass content analysis are two important factors that contribute to LMWH quality control. Size exclusion chromatography (SEC), relying on multiple angle laser scattering (MALS)/refractive index (RI) detectors, has been developed for accurate analysis of heparin MW in the absence of standards. However, the cations, which ion-pair with the anionic polysaccharide chains of heparin and LMWHs, had not been considered in previous reports. In this study, SEC with MALS/RI and inductively coupled plasma/mass spectrometry detectors were used in a comprehensive analytical approach taking both anionic polysaccharide and ion-paired cations heparin products. This approach was also applied to quantitative analysis of heparin and LMWHs. Full profiles of MWs and mass recoveries for three commercial heparin/LMWH products, heparin sodium, enoxaparin sodium and nadroparin calcium, were obtained and all showed higher MWs than previously reported. This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides.

•Both O- and N-glycans on the rhiBSP are enriched in sialic acids at their termini.•Four new N-glycosylation sites (N104, N177, N182, N190) of rhiBSP were identified.•SAs at both N- and O-glycans are important for BSP regulation of osteogenesis.•BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression.The highly glycosylated bone sialoprotein (BSP) is an abundant non-collagenous phosphoprotein in bone which enhances osteoblast differentiation and new bone deposition in vitro and in vivo. However, the structural details of its different glycosylation linkages have not been well studied and their functions in bone homeostasis are not clear. Previous studies suggested that the O-glycans, but not the N-glycans on BSP, are highly sialylated. Herein, we employed tandem mass spectrometry (MS/MS) to demonstrate that the N-glycanson the recombinant human integrin binding sialoprotein (rhiBSP) are also enriched in sialic acids (SAs) at their termini. We also identified multiple novel sites of N-glycan modification. Treatment of rhiBSP enhances osteoblast differentiation and mineralization of MC3T3-E1 cells and this effect could be partially reversed by efficient enzymatic removal of its N-glycans. Removal of all terminal SAs has a greater effect in reversing the effect of rhiBSP on osteogenesis, especially on mineralization, suggesting that sialylation at the termini of both N-glycans and O-glycans plays an important role in this regulation. Moreover, BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression. Collectively, our results identified novel N-glycans enriched in SAs on the rhiBSP and demonstrated that SAs at both N- and O-glycans are important for BSP regulation of osteoblast differentiation and mineralization in vitro.

•HPAEC-PAD-MS/MS was applied to sequence β-glucan extracted from highland barley.•G1 → 3G1 → 4G and G1 → 3G1 → 4G1 → 4G are the major repeating blocks of this β-glucan.•They were interspersed with homogeneous 1 → 4 linked domains with different lengths.•The ratio of 1 → 3 and 1 → 4 linkages in the β-glucan was approximately 1:3.Highland barley is one of the hull-less barleys that contains high amounts of β-glucan. A detailed sequence analysis is needed to support the application of the β-glucan from highland barley. The β-conformation and 1 → 3/1 → 4 linkages were confirmed using nuclear magnetic resonance (NMR) spectroscopy. Monosaccharide analysis was accomplished with high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) and showed that glucose (G) was the dominant sugar component of this β-glucan. HPAEC-PAD was applied with on-line electrospray ion - to quadrupole time – of – flight mass spectrometry (Q/TOF-MS) to sequence oligosaccharides enzymatically derived from this β-glucan. More than 20 oligosaccharides were observed in the partially digested β-glucan mixture and their degree of polymerization (dp), confirmed by MS analysis, ranged from dp 2 to dp 20. The sequences of oligosaccharide dp2 – dp7 in the digested product mixture were unambiguously determined with MS/MS. Based on the sequence analysis of these oligosaccharides, G1→3G1→4G and G1→3G1→4G1→4G were determined to be the major repeating blocks of this β-glucan, and they were interspersed with homogeneous 1 → 4 linked domains with different lengths. The ratio of 1 → 3 and 1 → 4 linkages in the β-glucan was approximately 1:3.Download high-res image (192KB)Download full-size image

The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities. This work describes a strategy that combines an efficient release, labeling and liquid chromatography-mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry (MS) analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using GlycResoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains >8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.

•The factors impact on the degree of oxidation of starch were examined.•The new structures were elucidated carefully in the TEMPO-NaBr-NaClO reactions.•Specific oxidation pattern of starch with TEMPO-NaBr-NaClO was demonstrated.Oxidized starch, one of the most important starch derivatives, has many different properties and applications. Currently, there are two ways to produce oxidized starch, through specific and nonspecific oxidation. Specific oxidation using the stable nitroxyl radical, 2,2,6,6-tetramethyl preparidinloxy (TEMPO), with NaBr and NaClO can produce oxidized starches with different properties under good quality control. In the current study, we examine the products of specifically oxidized starch. As the amount of oxidant and the temperature, two critical factors impacting the oxidation of starch were thoroughly investigated. Analysis of the molecular weight (MW), degree of oxidization (DO) and the detailed structures of corresponding products was accomplished using gel permeation chromatography with multi-angle laser light scattering (GPC-MALLS), infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and quadrapole time-of-flight mass spectrometry (Q/TOF-MS). According to the analytical results, the oxidation patterns of starch treated with specific oxidant TEMPO-NaBr-NaClO were established. When high amounts of oxidant was applied, more glucose residues within starch were oxidized to glucuronic acids (higher DO) and substantial degradation to starch oligosaccharides was observed. By selecting a reaction temperature of 25 °C a high DO could be obtained for a given amount of oxidant. The reducing end sugar residue within oxidized starch was itself oxidized and ring opened in all TEMPO-NaBr-NaClO reactions. Furthermore, extra oxidant generated additional novel structures in the reducing end residues of some products, particularly in low temperature reactions.

Low molecular weight heparins (LMWHs) are polydisperse and microheterogenous mixtures of polysaccharides used as anticoagulant drugs. Profiling analysis is important for obtaining deeper insights into the structure of LMWHs. Previous oligosaccharide mapping methods are relatively low resolution and are unable to show an entire picture of the structural complexity of LMWHs. In the current study a profiling method was developed relying on multiple heart-cutting, two-dimensional, ultrahigh performance liquid chromatography with quadruple time-of-flight mass spectrometry. This represents an efficient, automated, and robust approach for profiling LMWHs. Using size-exclusion chromatography and ion-pairing reversed-phase chromatography in a two-dimensional separation, LMW components of different sizes and LMW components of the same size but with different charges and polarities can be resolved, providing a more complete picture of a LMWH. Structural information on each component was then obtained with quadrupole time-of-flight mass spectrometry. More than 80 and 120 oligosaccharides were observed and unambiguously assigned from the LMWHs, nadroparin and enoxaparin, respectively. This method might be useful for quality control of LMWHs and as a powerful tool for heparin-related glycomics.

•This work provide a method with IC hyphenated MS and PAD in parallel.•This method was developed to analyze oligosaccharides.•The types of branches in Dextran were identified with IC-MS/MS.•The degree of each type of branch in Dextran was measured with IC-PAD.•This method provides qualitative and quantitative analysis of carbohydrates.Dextran, a family of natural polysaccharides, consists of an α (1→6) linked-glucose main (backbone) chain having a number of branches. The determination of the types and the quantities of branches in dextran is important in understanding its various biological roles. In this study, a hyphenated method using high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to qualitative and quantitative analysis of dextran branches. A rotary cation-exchange cartridge array desalter was used for removal of salt from the HPAEC eluent making it MS compatible. MS and MS/MS were used to provide structural information on the enzymatically prepared dextran oligosaccharides. PAD provides quantitative data on the ratio of enzyme-resistant, branched dextran oligosaccharides. Both the types and degree of branching found in a variety of dextrans could be simultaneously determined online using this method.

Alginate is a linear and acidic polysaccharide, composed of (1 → 4) linked β-d-mannuronic acid (ManA) and α-l-guluronic acid (GulA). The ratio of ManA to GulA (M/G) is one of the most important factors for the application of alginate and its derivatives in various areas. In this work, a robust and accurate method was developed to analyze M/G using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The impact of hydrolysis conditions on the release patterns of ManA and GulA from alginate and its derivatives was investigated. The release patterns of ManA and GulA need to be considered separately to obtain an accurate M/G. Several hydrolysis conditions were established that released ManA and GulA completely and maintained these saccharide residues intact. The proper M/G of alginates from different sources and its derivatives could then be calculated by integration of the corresponding ManA and GulA peaks.

Investigation of the action pattern of enzymes acting on carbohydrates is challenging, as both the substrate and the digestion products are complex mixtures. Dextran and its enzyme-derived oligosaccharides are widely used for many industrial applications. In this work, a new method relying on ultra-performance hydrophilic interaction liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UP-HILIC- Q/TOF-MS/MS) was developed to analyze a complex mixture of dextran oligosaccharide products to determine the action pattern of dextranase. No derivatization of oligosaccharides was required and the impact of the α- and β-configurations of the native oligosaccharides on the chromatographic separation was eliminated. The 1→6, 1→3, 1→4 backbone linkages and the branch linkages of these oligosaccharides were all distinguished from diagnostic ions in their MS/MS spectra, including fragments corresponding to 0,2A, 0,3A, 0,4A, B-H2O, 2,5A, and 3,5A. The sequences of the oligosaccharide products were similarly established. Thus, the complex oligosaccharide mixtures in dextran digestion products were profiled and identified using this method. The more enzyme-resistant structures in dextran were established using much less sample, labor, time, and uncertainty than in previous studies. This method provides an efficient, sensitive, and straightforward way to monitor the entire process of digestion, establish the action pattern of the dextranase from Penicillium sp., and to support the proper industrial application of dextranase.

The sulfo groups of glycosaminoglycans contribute to their high charge densities, and are critical for the role they play in various physiological and pathophysiological processes. Unfortunately, the sulfo groups can be hydrolyzed to inorganic sulfate. Thus, it is important to monitor the presence of these sulfo groups. In addition, free anions, including chloride, sulfate and acetate, are often present in glycosaminoglycans as a result of multiple purification steps, and their presence also needs to be monitored. In this report, ion chromatography with conductivity detection is used to analyze the anions present in glycosaminoglycans, including heparin, heparan sulfate, chondroitin sulfate and dermatan sulfate. This method allows quantitation over a wide range of concentrations, affording a limit of quantitation of 0.1 ppm and a limit of detection of 0.05 ppm for most anions of interest. The stability of heparin was also studied, providing data on the formation of both sulfate and acetate anions.

•New insights into action patterns of two lyases were taken.•HA oligosaccharide products having both even & odd-number residues were observed.•Chondroitinase AC I acted more efficiently at the β 1–4 glycosidic bond of HA.•Hyaluronidase cleaves both β 1–4 and β 1–3 glycosidic linkages of HA.Hyaluronic acid (HA), a glycosaminoglycan, is a linear polysaccharide with negative charge, composed of a repeating disaccharide unit [→4)-β-d-glucopyranosyluronic acid (1 → 3)-β-d- N-acetyl-d-glucoaminopyranose (1 → ]n ([→4) GlcA (1 → 3) GlcNAc 1 → ]n). It is widely used in different applications based on its physicochemical properties associated with its molecular weight. Enzymatic digestion by polysaccharides lyases is one of the most important ways to decrease the molecular weight of HA. Thus, it is important to understand the action patterns of lyases acting on HA. In this study, the action patterns of two common lyases, Flavobacterial chondroitinase AC I and Streptomyces hyaluronidase, were investigated by analyzing HA oligosaccharide digestion products. HA oligosaccharides having an odd-number of saccharide residues were observed in the products of both lyases, but their distributions were quite different. Chondroitinase AC acted more efficiently at the β 1–4 glycosidic bond linking GlcNAc and GlcA. Oligosaccharides, having an even number of saccharide residues, and with an unsaturated uronic acid (4-deoxy-α-l-threo-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end represent the major product. A minor amount of oligosaccharides having an odd number of saccharide residues resulted from the irregular terminal residues of HA substrate chains. Hyaluronidase showed a more complicated product mixture. Its minimum recognition and digestion domain is HA heptasaccharide and it could cleave both β 1–4 and β 1–3 glycosidic linkages. The HA oligosaccharides, generated with a 2-acetamido-2,3-di-deoxy-β-d-erythro-hex-2-enopyranose (△HexNAc) at the non-reducing end, are believed to be unstable and undergo breakdown immediately after their generation, and the oligosaccharides with △UA residue at the non-reducing end are formed. Thus, oligosaccharides having both an even and odd-number saccharide residues with a △UA residue at their non-reducing ends, represent the major products of hyaluronidase acting on HA.