Top-Down Approach for the Direct Characterization of Low Molecular Weight Heparins Using LC-FT-MS

Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy

Complete Mass Spectral Characterization of a Synthetic Ultralow-Molecular-Weight Heparin Using Collision-Induced Dissociation

Glycosaminoglycans (GAGs) are a class of biologically important molecules, and their structural analysis is the target of considerable research effort. Advances in tandem mass spectrometry (MS/MS) have recently enabled the structural characterization of several classes of GAGs; however, the highly sulfated GAGs, such as heparins, have remained a relatively intractable class due their tendency to lose SO₃ during MS/MS, producing few sequence-informative fragment ions. The present work demonstrates for the first time the complete structural characterization of the highly sulfated heparin-based drug Arixtra. This was achieved by Na⁺/H⁺ exchange to create a more ionized species that was stable against SO₃ loss, and that produced complete sets of both glycosidic and cross-ring fragment ions. MS/MS enables the complete structural determination of Arixtra, including the stereochemistry of its uronic acid residues, and suggests an approach for solving the structure of more complex, highly sulfated heparin-based drugs

FGF–FGFR Signaling Mediated through Glycosaminoglycans in Microtiter Plate and Cell-Based Microarray Platforms

Fibroblast growth factor (FGF) signals cell growth through its interaction with a fibroblast growth factor receptor (FGFR) and a glycosaminoglycn (GAG) coreceptor. Here, we examine the signaling of five different FGFs (1, 2, 6, 8, and 8b) through FGFR3c. A small library of GAG and GAG-derivative coreceptors are screened to understand better the structure–activity relationship of these coreceptors on signaling. Initially, data were collected in a microtiter plate well-based cell proliferation assay. In an effort to reduce reagent requirements and improve assay throughput, a cell-based microarray platform was developed. In this cell-based microarray, FGFR3c-expressing cells were printed in alginate hydrogel droplets of ∼30 nL and incubated with FGF and GAG. Heparin was the most effective GAG coreceptor for all FGFs studied. Other GAGs, such as 2-<i>O</i>-desulfated heparin and chondroitin sulfate B, were also effective coreceptors. Signaling by FGF 8 and FGF 8b showed the widest tolerance for coreceptor structure. Finally, this on-chip cell-based microarray provides comparable data to a microtiter plate well-based assay, demonstrating that the coreceptor assay can be converted into a high-throughput assay

Glycosaminoglycanomics of Cultured Cells Using a Rapid and Sensitive LC-MS/MS Approach

Glycosaminoglycans (GAGs), a family of polysaccharides widely distributed in eukaryotic cells, are responsible for a wide array of biological functions. Quantitative disaccharide compositional analysis is one of the primary ways to characterize the GAG structure. This structural analysis is typically time-consuming (1–2 weeks) and labor intensive, requiring GAG recovery and multistep purification, prior to the enzymatic/chemical digestion of GAGs, and finally their analysis. Moreover, 10⁵–10⁷ cells are usually required for compositional analysis. We report a sensitive, rapid, and quantitative analysis of GAGs present in a small number of cells. Commonly studied cell lines were selected based on phenotypic properties related to the biological functions of GAGs. These cells were lysed using a commercial surfactant reagent, sonicated, and digested with polysaccharide lyases. The resulting disaccharides were recovered by centrifugal filtration, labeled with 2-aminoacridone, and analyzed by liquid chromatography (LC)-mass spectrometry (MS). Using a highly sensitive MS method, multiple reaction monitoring (MRM), the limit of detection for each disaccharide was reduced to 0.5–1.0 pg, as compared with 1.0–5.0 ng obtained using standard LC-MS analysis. Sample preparation time was reduced to 1–2 days, and the cell number required was reduced to 5000 cells for complete GAG characterization to as few as 500 cells for the characterization of the major GAG disaccharide components. Our survey of the glycosaminoglycanomes of the 20 selected cell lines reveals major differences in their GAG amounts and compositions. Structure–function relationships are explored using these data, suggesting the utility of this method in cellular glycobiology

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

<p>A β-glucosidase extracted from bitter almond (<i>Prunus dulcis var. amara</i>) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% <i>t</i>-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (<i>V</i>₀) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219 mM and 116 mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336 h of reaction, the OG yield and the conversion rate of glucose reached 134 mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.</p

Composition of Glycosaminoglycans in Elasmobranchs including Several Deep-Sea Sharks: Identification of Chondroitin/Dermatan Sulfate from the Dried Fins of <i>Isurus oxyrinchus</i> and <i>Prionace glauca</i>

<div><p>Shark fin, used as a food, is a rich source of glycosaminoglyans (GAGs), acidic polysaccharides having important biological activities, suggesting their nutraceutical and pharmaceutical application. A comprehensive survey of GAGs derived from the fin was performed on 11 elasmobranchs, including several deep sea sharks. Chondroitin sulfate (CS) and hyaluronic acid (HA) were found in <i>Isurus oxyrinchus</i>, <i>Prionace glauca</i>, <i>Scyliorhinus torazame</i>, <i>Deania calcea</i>, <i>Chlamydoselachus anguineus</i>, <i>Mitsukurina owatoni</i>, <i>Mustelus griseus</i> and <i>Dasyatis akajei</i>, respectively. CS was only found from <i>Chimaera phantasma</i>, <i>Dalatias licha</i>, and <i>Odontaspis ferox</i>, respectively. Characteristic disaccharide units of most of the CS were comprised of C- and D-type units. Interestingly, substantial amount of CS/dermatan sulfate (DS) was found in the dried fin (without skin and cartilage) of <i>Isurus oxyrinchus</i> and <i>Prionace glauca</i>. ¹H-NMR analysis showed that the composition of glucuronic acid (GlcA) and iduronic acid (IdoA) in shark CS/DS was 41.2% and 58.8% (<i>Isurus oxyrinchus</i>), 36.1% and 63.9% (<i>Prionace glauca</i>), respectively. Furthermore, a substantial proportion of this CS/DS consisted of E-, B- and D-type units. Shark CS/DS stimulated neurite outgrowth of hippocampal neurons at a similar level as DS derived from invertebrate species. Midkine and pleiotrophin interact strongly with CS/DS from <i>Isurus oxyrinchus</i> and <i>Prionace glauca</i>, affording <i>K_d</i> values of 1.07 nM, 6.25 nM and 1.70 nM, 1.88 nM, respectively. These results strongly suggest that the IdoA-rich domain of CS/DS is required for neurite outgrowth activity. A detailed examination of oligosaccharide residues, produced by chondroitinase ACII digestion, suggested that the IdoA and B-type units as well as A- and C-type units were found in clusters in shark CS/DS. In addition, it was discovered that the contents of B-type units in these IdoA-rich domain increased in a length dependent manner, while C- and D-type units were located particularly in the immediate vicinity of the IdoA-rich domain.</p></div

Determination of unsaturated disaccharide components in IdoA-rich fractions of different molecular weights.

<p>(A) Separation of IdoA-rich domain and partially degraded samples by ChaseACII. One mg of samples were subjected partial digestion with 1 unit of ChaseACII at 37°C for 1 h and then resulting degraded samples were fractionated by an HPSEC systems with an Asahipak 510HQ column (7.6 mm, i.d. × 300 mm) as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120860#pone.0120860.ref027" target="_blank">27</a>]. The fraction consisting of oligosaccharides contained an IdoA-rich domain as the GlcA-rich domains were degraded by ChaseACII. (B) Separation of IdoA-rich domain at the different molecular weight. Samples (0.1 mg) after digestion with ChaseACII were subjected by gradient SDS-PAGE (PAGEL NPG-1020L, 10–20%). The electrophoresis was performed as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120860#pone.0120860.ref027" target="_blank">27</a>]. (C) Determination of unsaturated disaccharides in IdoA-rich domain at the different molecular weight. (D) Determination of unsaturated disaccharides in fraction b, c and d in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120860#pone.0120860.g006" target="_blank">Fig. 6A</a>. Peaks: 1, ΔDi-0S; 2, ΔDi-4S; 3, ΔDi-6S; 4, ΔDiUA-2S; 5, ΔDi-diS_E; 6, ΔDi-diS_B; 7, ΔDi-diS_D.</p

Binding of CS/DS from shortfin mako shark (Fr. 5) and blue shark (Fr. 3) to immobilize to growth factors.

<p>Various concentrations of shark CS/DS and squid CS-E (Seikagaku Corp., Tokyo, Japan) were injected onto the surface of a pleiotrophin- or midkine-immobilized sensor tip. Sensorgrams obtained with various concentrations of each shark CS/DS were evaluated using BIAevaluation 3.0 software. RU, resonance units.</p

Effect of shark CS/DS from shortfin mako shark (Fr. 5) and blue shark (Fr. 3) on neurite outgrowth.

<p>(A) Representative morphological features of E16 hippocampal neurons cultured with shark CS/DS. E16 hippocampal neuronal cells (16000 cells/cm²) were cultured for 18h on various substrates coated on P-ORN, fixed and immunostained as described under “Materials and Methods”. (B) The mean length of the longest neurite was measured for more than 50 randomly selected neurons cultured on various substrates (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120860#sec002" target="_blank">Materials and Methods</a>”). The values obtained from six independent experiments are expressed as the mean ±S.E. Mann-Whitney’s U test was used to evaluate the significance of differences between means (**, <i>p</i><0.01).</p

Bottom-Up Low Molecular Weight Heparin Analysis Using Liquid Chromatography-Fourier Transform Mass Spectrometry for Extensive Characterization

Low molecular weight heparins (LMWHs) are heterogeneous, polydisperse, and highly negatively charged mixtures of glycosaminoglycan chains prescribed as anticoagulants. The detailed characterization of LMWH is important for the drug quality assurance and for new drug research and development. In this study, online hydrophilic interaction chromatography (HILIC) Fourier transform mass spectrometry (FTMS) was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase II digestion. More than 40 oligosaccharide fragments of LMWH were quantified and used to compare LMWHs prepared by three different manufacturers. The quantified fragment structures included unsaturated disaccharides/oligosaccharides arising from the prominent repeating units of these LMWHs, 3-<i>O</i>-sulfo containing tetrasaccharides arising from their antithrombin III binding sites, 1,6-anhydro ring-containing oligosaccharides formed during their manufacture, saturated uronic acid oligosaccharides coming from some chain nonreducing ends, and oxidized linkage region oligosaccharides coming from some chain reducing ends. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility. When combined with the top-down approach, HILIC LC-FTMS based analysis should be suitable for the advanced quality control and quality assurance in LMWH production