Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

Henriksen, Jens H.; Roepstorff, Peter; Ringborg, Lene Hoffmeyer

doi:10.1016/j.carres.2005.11.030

Cited by 46 publications

(43 citation statements)

References 24 publications

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“…In the case of heparin hexamers, only two masses are required to fit the experimental data using eq 1, 1651 ( 2 Da and 1731 ( 2 Da, which correspond to heparin fragments (6,8,0) and (6,9,0). Another abundant hexamer, (6, 7, 0) does not appear to bind to the protein under the conditions tested.…”

Section: Resultsmentioning

confidence: 99%

Glycosaminoglycans as Naturally Occurring Combinatorial Libraries: Developing a Mass Spectrometry-Based Strategy for Characterization of Anti-Thrombin Interaction with Low Molecular Weight Heparin and Heparin Oligomers

2007

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Heparin is a densely charged polysaccharide, which is best known for its anticoagulant activity, although it also modulates a plethora of other biological processes. Unlike biopolymers whose synthesis is strictly controlled by a unique genetic template, heparin molecules exhibit a remarkable degree of structural heterogeneity, which poses a serious challenge for studies of heparin-protein interactions. This analytical challenge is often dealt with by reducing the enormous structural repertoire of heparin to a model small molecule. In this paper, we describe a different approach inspired by the experimental methodologies from the arsenal of combinatorial chemistry. Interaction of anti-thrombin III (AT) with heparinoids is studied using a mixture of oligoheparin molecules of fixed degree of polymerization, but varying chemical composition (heparin hexasaccharides obtained by size exclusion chromatography of an enzymatic digest of porcine intestinal heparin with bacterial heparinase), as well as a heparin-derived pharmaceutical preparation Tinzaparin (heparin oligosaccharides up to a 22-mer). AT binders are identified based on the results of ESI MS measurements of complexes formed by protein-oligoheparin association. Additionally, differential depletion of free heparin oligomers in solution in the presence of AT is used to verify the binding preferences. ESI MS characterization of oligoheparin-AT interaction under partially denaturing conditions allowed the conformer specificity of the protein-polyanion binding to be monitored. A model emerging from these studies invokes the notion of a well-defined binding site on AT, to which a flexible partner (heparin) adapts to maximize favorable intermolecular electrostatic interactions. This study demonstrates the enormous potential of ESI MS as an analytical tool to study the interactions of highly heterogeneous glycosaminoglycans with their cognate proteins outside of the commonly accepted reductionist paradigm, which reduces the intrinsic complexity of heparin by using structurally defined homogeneous low molecular weight mimetics.

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Section: Resultsmentioning

confidence: 99%

Glycosaminoglycans as Naturally Occurring Combinatorial Libraries: Developing a Mass Spectrometry-Based Strategy for Characterization of Anti-Thrombin Interaction with Low Molecular Weight Heparin and Heparin Oligomers

2007

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“…For complex mixtures, the spectrum might, therefore, become impossible to interpret due to the overlapping of isobaric species. For these reasons, the structural analysis of complex GAG mixtures claimed lately for the combination of MS with efficient separation techniques such as capillary electrophoresis [19,20,22,23], high-performance liquid chromatography [13,24], high-performance cation exchange [25], or size exclusion chromatography [26]. These strategies, however, include several analytical steps such as separation, MS screening, and sequencing of the GAG species in on-line, on-the-fly tandem MS [10], entailing specially designed interfaces and laborious optimization procedures.…”

mentioning

confidence: 99%

Analysis of oversulfation in a chondroitin sulfate oligosaccharide fraction from bovine aorta by nanoelectrospray ionization quadrupole time-of-flight and fourier-transform ion cyclotron resonance mass spectrometry

Mormann

Zamfir

Seidler

et al. 2007

J. Am. Soc. Mass Spectrom.

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A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B-and C-type ions and their respective complementary Y-and Z-type fragment ions. (J Am Soc Mass Spectrom 2007, 18, 179 -187)

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“…This reduction in "chemical noise" allowed for simplified and interpretable spectra [45]. On-line cation exchange has also been demonstrated to enable efficient ionization of heparin oligosaccharides by preventing the IPR from entering the mass spectrometer [46]. Kuberan et al studied ionization efficiencies of different volatile IPRs for coupling of RPIP-HPLC to ESI-MS. Dibutylammonium (DBA) acetate in a methanol gradient proved to be successful in separating heparin oligosaccharides on a C 18 column with detection and analysis by ESI-TOF-MS [47].…”

Section: Rp Ion-pairmentioning

confidence: 98%

Ion chromatography for the separation of heparin and structurally related glycoaminoglycans: A review

Fasciano

Danielson

2016

J of Separation Science

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The global crisis resulting from adulterated heparin in late 2007 and early 2008 revived the importance of analytical techniques for the purity analysis of heparin products. The utilization of ion chromatography techniques for the separation, detection, and structural determination of heparin and structurally related glycoaminoglycans, including their corresponding oligosaccharides, has become increasingly important. This review summarizes the primary HPLC approaches, particularly strong anion exchange, weak ion exchange, and reversed-phase ion-pair, used for heparin purity analysis as well as structural characterization. Strong anion exchange HPLC has been studied most extensively and currently offers the best separation of crude heparin and heparin-like compounds. Weak anion exchange HPLC has been shown to provide shorter analysis times with lower salt concentrations in the mobile phase but is not as widely developed for the separation of all glycoaminoglycans of interest. Reversed-phase ion-pair HPLC offers fast and effective separations of oligosaccharides derived from glycoaminoglycans that can be coupled to mass spectrometry for structural analysis. However, this method generally does not provide sufficient retention of intact glycoaminoglycans.

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Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

Cited by 46 publications

References 24 publications

Glycosaminoglycans as Naturally Occurring Combinatorial Libraries: Developing a Mass Spectrometry-Based Strategy for Characterization of Anti-Thrombin Interaction with Low Molecular Weight Heparin and Heparin Oligomers

Glycosaminoglycans as Naturally Occurring Combinatorial Libraries: Developing a Mass Spectrometry-Based Strategy for Characterization of Anti-Thrombin Interaction with Low Molecular Weight Heparin and Heparin Oligomers

Analysis of oversulfation in a chondroitin sulfate oligosaccharide fraction from bovine aorta by nanoelectrospray ionization quadrupole time-of-flight and fourier-transform ion cyclotron resonance mass spectrometry

Ion chromatography for the separation of heparin and structurally related glycoaminoglycans: A review

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