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Nicola Volpi - One of the best experts on this subject based on the ideXlab platform.
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novel reverse phase ion pair high performance liquid chromatography separation of heparin heparan sulfate and low molecular weight heparins Disaccharides and oligosaccharides
Journal of Chromatography A, 2013Co-Authors: Fabio Galeotti, Nicola VolpiAbstract:Abstract In this study, by using tetrabutylammonium bisulfate as ion-pairing reagent, we were able to separate all the main heparin/heparan sulfate Disaccharides generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit and resistant to enzymatic action. Moreover, this novel HPLC method was able to separate and quantify uncommon Disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity ∼4-times higher compared to conventional strong-anion exchange-HPLC separation. This was obtained by a common UV detector at 232 nm avoiding the use of complex procedures capable of increasing sensitivity by post-column derivatization. Finally, it is worth mentioning that disaccharide/oligosaccharide composition by HPLC and UV detection is a common analytical approach in quality control laboratories to evaluate heparins and low molecular weight-heparins structure and quality during their extraction and production. This simple HPLC approach offers high resolution and sensitivity for the rapid differentiation of pharmaceutical native heparins and derivatives and for the compositional analysis of small amounts of samples derived from biological sources at a glycosaminoglycans level of a few hundred nanogram.
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Structural characterisation of chondroitin sulphate from Italian cheese Parmigiano-Reggiano
Food Chemistry, 2012Co-Authors: Giovanni V. Coppa, Francesca Maccari, Lucia Zampini, Lucia Santoro, Tiziana Galeazzi, Orazio Gabrielli, Nicola VolpiAbstract:abstract Chondroitin sulphate (CS) was purified for the first time from the Italian cheese Parmigiano-Reggiano andanalysed to evaluate its structure and properties. Two main polysaccharides were identified as CS, 72%,and fast moving-heparin/heparan sulphate, 28%. Quantitative analyses yielded 1.5–3.0 l g of totalGAGs per gram of Parmigiano-Reggiano (0.15–0.30%). By means of specific chondroitinases and HPLCseparation of generated unsaturated Disaccharides, CS was found to be composed of 9% of nonsulphateddisaccharide, 26% of disaccharide monosulphated in 6 of the GalNAc, 65% of disaccharide monosulph-ated in 4 of the GalNAc, with a charge density of 0.91 and a 4/6-sulphated ratio of 2.45. The ratio of 4/6sulphated residues was confirmed by 13 C NMR experiments. Susceptibility to cleavage catalysed by chon-droitinase B confirmed that the purified Parmigiano-Reggiano CS contains mainly GlcA ( 94%) as uronicacid. PAGE analysis showed a CS having a molecular mass with an average value of 15400. 2012 Elsevier Ltd. All rights reserved.
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online reverse phase high performance liquid chromatography fluorescence detection electrospray ionization mass spectrometry separation and characterization of heparan sulfate heparin and low molecular weight heparin Disaccharides derivatized with 2
Analytical Chemistry, 2011Co-Authors: Fabio Galeotti, Nicola VolpiAbstract:A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of Disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated Disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent Disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ∼15–21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of ...
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high performance liquid chromatography and on line mass spectrometry detection for the analysis of chondroitin sulfates hyaluronan Disaccharides derivatized with 2 aminoacridone
Analytical Biochemistry, 2010Co-Authors: Nicola VolpiAbstract:Abstract In this study, we developed an on-line reverse-phase high-performance liquid chromatography–electrospray ionization–mass spectrometry (RP–HPLC–ESI–MS) separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) Disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC), providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI–MS spectral acquisition. Isomeric nonsulfated HA and CS/DS Disaccharides, isomeric monosulfated and isomeric disulfated CS/DS Disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged Disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP–HPLC–ESI–MS of each AMAC disaccharide was found to be linear from 3 to 500 ng with very high coefficient of correlation values due to the high efficiency of separation and the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition, and minor oligomer species identified as GalNAcSO4 at the nonreducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS Disaccharides were also evaluated by means of RP–HPLC–(Fp)–ESI–MS.
Julie A Leary - One of the best experts on this subject based on the ideXlab platform.
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a comprehensive compositional analysis of heparin heparan sulfate derived Disaccharides from human serum
Analytical Chemistry, 2011Co-Authors: Milady R Ninonuevo, Anish Sharma, Lieza M Dananleon, Julie A LearyAbstract:The analysis of heparan sulfate glycosaminoglycans (HSGAGs) variations in human serum at the disaccharide level has a great potential for disease diagnosis and prognosis. However, the lack of available analytical methodology for the compositional analysis of HSGAGs in human serum remains to be addressed to delineate the possible role of HSGAGs on the onset and/or progression of a disease. In this study, we have developed a method for the in-depth compositional analysis of the 12 heparin/HS-derived Disaccharides from human serum using a combination of technologies – fractionation, exhaustive digestion, solid phase extraction and LC-MS/MS. The method exhibits high recovery (72%–110%) and good reproducibility (standard deviation of less than 5%) with a low limit of detection and quantification. Errors from the method validation were within 1.1%. Non-detectable non- or low-sulfated Disaccharides in human serum were also detected using the optimized protocol. Further applying this method, the comprehensive analysis of HSGAGs compositions in human sera from female donors showed considerable variations in disaccharide patterns and compositions.
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detection and quantification of the sulfated Disaccharides in chondroitin sulfate by electrospray tandem mass spectrometry
Journal of the American Society for Mass Spectrometry, 2000Co-Authors: Heather Desaire, Julie A LearyAbstract:A new method of identifying and quantifying the disaccharide building blocks of glycosaminoglycans is introduced. The polysaccharides are subjected to an enzymatic digestion that releases the sulfated Disaccharides. The Disaccharides are then identified using a combination of electrospray ionization mass spectrometry and tandem mass spectrometry. Quantification of the isomeric Disaccharides is also achieved by tandem mass spectrometry, using a recently developed methodology which quantifies mixtures of isomers without the use of chromatography or prior separation. Using mass spectrometry to characterize the components of glycosaminoglycans significantly reduces both sample consumption and analysis time of traditional methods.
Robert J Linhardt - One of the best experts on this subject based on the ideXlab platform.
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analysis of total human urinary glycosaminoglycan Disaccharides by liquid chromatography tandem mass spectrometry
Analytical Chemistry, 2015Co-Authors: Lingyun Li, Katherine H Overdier, Lee Anne Ammons, Ivor S Douglas, Clay Cothren Burlew, Fuming Zhang, Eric P Schmidt, Robert J LinhardtAbstract:The determination of complex analytes, present at low concentrations, in biological fluids poses a difficult challenge. This study relies on an optimized method of recovery, enzymatic treatment, and disaccharide analysis by liquid chromatography–tandem mass spectrometry to rapidly determine low concentrations of glycosaminoglycans in human urine. The approach utilizes multiple reaction monitoring (MRM) of glycosaminoglycan Disaccharides obtained from treating urine samples with recombinant heparin lyases and chondroitin lyase. This rapid and sensitive method allows the analysis of glycosaminoglycan content and disaccharide composition in urine samples having concentrations 10- to 100-fold lower than those typically analyzed from patients with metabolic diseases, such as mucopolysaccharidosis. The current method facilitates the analysis low (ng/mL) levels of urinary glycosaminoglycans present in healthy individuals and in patients with pathological conditions, such as inflammation and cancers, that can sub...
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impact of salt exposure on n acetylgalactosamine 4 sulfatase arylsulfatase b activity glycosaminoglycans kininogen and bradykinin
Glycoconjugate Journal, 2013Co-Authors: Kumar Kotlo, Leo Feferman, Sumit Bhattacharyya, Shah Tejaskumar, Robert S Danziger, Robert J Linhardt, Bo Yang, Joanne K TobacmanAbstract:N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S Disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived Disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.
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Capillary Electrophoresis for the Analysis of Glycosaminoglycan-Derived Disaccharides
Methods of Molecular Biology, 2013Co-Authors: Yuqing Chang, Bo Yang, Amanda Weyers, Robert J LinhardtAbstract:Capillary electrophoresis is a common technique used for glycosaminoglycan-derived disaccharide analysis because of its high resolving power, high separation efficiency, high sensitivity, short analysis time, and straightforward operation. CE coupled to laser-induced fluorescence (LIF) detection shows an approximately 100 times higher sensitivity than traditional UV detection at 232 nm. 2-Aminoacridone (AMAC) is a widely used fluorophore for labeling unsaturated Disaccharides by deductive amination, which is one of the most important method of derivatization of Disaccharides for CE-LIF detection. Outlined in this chapter is a protocol of analyzing glycosaminoglycan-derived Disaccharides by CE-LIF with AMAC derivatization.
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quantification of heparan sulfate Disaccharides using ion pairing reversed phase microflow high performance liquid chromatography with electrospray ionization trap mass spectrometry
Analytical Chemistry, 2009Co-Authors: Zhenqing Zhang, Robert J LinhardtAbstract:The glycosaminoglycan (GAG) family of biomacromolecules is composed acidic and linear chains of repeating disaccharide units. Quantitative disaccharide composition analysis is essential for the study and characterization of GAGs. Heparan sulfate and heparin consist of multiple disaccharide units and can be well-separated by ion-pairing reversed-phase microflow high-performance liquid chromatography (IPRP-Mf-HPLC). Each disaccharide can be detected and its mass confirmed by electrospray ionization mass spectrometry (ESI-MS). Isotopically enriched Disaccharides were prepared chemoenzymatically from a uniformly 13C,15N-labeled N-acetylheparosan (−GlcA(1→4)GlcNAc−) obtained from the fermentation of E. coli K5. These isotopically enriched Disaccharides have identical HPLC retention times and mass spectra as their unlabeled counterparts and were used in liquid chromatography−mass spectrometry (LC−MS) as internal standards. The ratio of intensities between each pair of enriched and nonenriched Disaccharides show...
Fabio Galeotti - One of the best experts on this subject based on the ideXlab platform.
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novel reverse phase ion pair high performance liquid chromatography separation of heparin heparan sulfate and low molecular weight heparins Disaccharides and oligosaccharides
Journal of Chromatography A, 2013Co-Authors: Fabio Galeotti, Nicola VolpiAbstract:Abstract In this study, by using tetrabutylammonium bisulfate as ion-pairing reagent, we were able to separate all the main heparin/heparan sulfate Disaccharides generated by the action of heparinases along with the main Hep tetrasaccharide possessing a 3-O-sulfate group on the sulfoglucosamine unit and resistant to enzymatic action. Moreover, this novel HPLC method was able to separate and quantify uncommon Disaccharides/oligosaccharides present in low molecular weight-heparins produced by chemical treatment with nitrous acid, dalteparin, or benzylation followed by alkaline hydrolysis, enoxaparin. Additionally, this procedure yields a sensitivity ∼4-times higher compared to conventional strong-anion exchange-HPLC separation. This was obtained by a common UV detector at 232 nm avoiding the use of complex procedures capable of increasing sensitivity by post-column derivatization. Finally, it is worth mentioning that disaccharide/oligosaccharide composition by HPLC and UV detection is a common analytical approach in quality control laboratories to evaluate heparins and low molecular weight-heparins structure and quality during their extraction and production. This simple HPLC approach offers high resolution and sensitivity for the rapid differentiation of pharmaceutical native heparins and derivatives and for the compositional analysis of small amounts of samples derived from biological sources at a glycosaminoglycans level of a few hundred nanogram.
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online reverse phase high performance liquid chromatography fluorescence detection electrospray ionization mass spectrometry separation and characterization of heparan sulfate heparin and low molecular weight heparin Disaccharides derivatized with 2
Analytical Chemistry, 2011Co-Authors: Fabio Galeotti, Nicola VolpiAbstract:A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of Disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated Disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent Disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ∼15–21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of ...
Kazuyuki Sugahara - One of the best experts on this subject based on the ideXlab platform.
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dermatan sulfate in tunicate phylogeny order specific sulfation pattern and the effect of 4idoa 2 sulfate β 1 3galnac 4 sulfate β 1 motifs in dermatan sulfate on heparin cofactor ii activity
BMC Biochemistry, 2011Co-Authors: Eliene O Kozlowski, P C Lima, Cristina P Vicente, Tito Monteiro Da Cruz Lotufo, Kazuyuki Sugahara, Mauro S G PavaoAbstract:Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated Disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated Disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated Disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units. Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.
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microanalysis of glycosaminoglycan derived oligosaccharides labeled with a fluorophore 2 aminobenzamide by high performance liquid chromatography application to disaccharide composition analysis and exosequencing of oligosaccharides
Analytical Biochemistry, 1999Co-Authors: Akiko Kinoshita, Kazuyuki SugaharaAbstract:Abstract A series of Disaccharides derived from chondroitin sulfate and heparin/heparan sulfate were derivatized at their reducing ends with a fluorophore 2-aminobenzamide to develop a sensitive microanalytical method for glycosaminoglycans. The resulting labeled compounds derived from chondroitin sulfate or heparin/heparan sulfate were well-separated and quantified by HPLC equipped with a fluorescence detector. The detection limit was a low picomole level. This method was applied to the analysis of the disaccharide composition of tetra- and hexasaccharides derived from chondroitin sulfate and heparin/heparan sulfate as well as these glycosaminoglycan polysaccharides. The method was also successfully applied to the exosequencing of chondrohexasaccharides, where the fluorophore-labeled oligosaccharides were degraded exolytically from the nonreducing ends using bacterial eliminases. The resultant labeled fragments were identified by HPLC.