Tandem MS can distinguish hyaluronic acid from <i>N</i>-acetylheparosan

Zhang, Zhenqing; Xie, Jin; Liu, Jian; Linhardt, Robert J.

doi:10.1016/j.jasms.2007.10.012

Cited by 22 publications

(15 citation statements)

References 43 publications

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“…Cross-ring fragmentation of the reducing end residue is observed by EID of 1 and 2 , but is not observed in EID of 3 , suggesting that the 1→3 glycosidic linkage inhibits this pathway of fragmentation. These observations are consistent with previous low-energy CAD studies which show that 1→4 linked HexNAc residues undergo facile retro-Diels-Alder reactions to yield 0,2 A products, but not 1→3 linked HexNAc residues [45,46], and similar results have been observed before for EDD of dermatan sulfate oligosaccharides [28], and for CAD of isobaric GAG oligosaccharides differing only in linkage position [47]. …”

Section: Resultssupporting

confidence: 92%

Electron-induced dissociation of glycosaminoglycan tetrasaccharides

Wolff

Laremore

Aslam

et al. 2008

J. Am. Soc. Mass Spectrom.

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Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a powerful tool for examining the structural features of sulfated glycosaminoglycans (GAGs). The characteristics of GAG fragmentation by EDD include abundant cross-ring fragmentation primarily on hexuronic acid residues, cleavage of all glycosidic bonds, and the formation of even-and odd-electron product ions. GAG dissociation by EDD has been proposed to occur through the formation of an excited species that can undergo direct decomposition or ejects an electron and then undergoes dissociation. In this work, we perform electron-induced dissociation (EID) on singly charged GAGs to identify products that form via direct decomposition by eliminating the pathway of electron detachment. EID of GAG tetra saccharides produces cleavage of all glycosidic bonds and abundant cross-ring fragmentation primarily on hexuronic acid residues, producing fragmentation similar to EDD of the same molecules, but distinctly different from the products of infrared multiphoton dissociation or collisionally activated decomposition. These results suggest that observed abundant fragmentation of hexuronic acid residues occurs as a result of their increased lability when they undergo electronic excitation. EID fragmentation of GAG tetrasaccharides results in both even-and odd-electron products. EID of heparan sulfate tetrasaccharide epimers produces identical fragmentation, in contrast to EDD, in which the epimers can be distinguished by their fragment ions. These data suggest that for EDD, electron detachment plays a significant role in distinguishing glucuronic acid from iduronic acid. [4][5][6]. GAGs are found both intra-and extracellularly in a wide variety of organisms, from bacteria to humans [7]. At the molecular level, GAGs are linear carbohydrates that consist of a repeating disaccharide containing a uronic acid (or hexose, in keratan sulfate) and a hexosamine. GAG complexity arises from variation in the degree and location of sulfation on either sugar, N-modification of the hexosamine, and C5 stereochemistry of the uronic acid. There is significant interest in developing methods to characterize the pattern of GAG modification because these structural details are believed to influence the biological function of GAGs. A wide variety of analytical techniques have been developed to characterize sulfated GAGs, including ID and Address reprint requests to Dr. L Jonathan Amster, University of Georgia, Department of Chemistry, Athens, GA 30602. E-mail: jamster@uga.edu 2D NMR [8] as well as mass spectrometry (MS) and tandem mass spectrometry (MS/MS) [9][10][11][12][13][14][15][16][17].The application of electron-ion interactions to MS/MS has produced a number of useful ion dissociation techniques. The recombination of low-energy electrons (::;1 eV) with multiply charged precursor ions, known as electron capture dissociation (ECD) [18], has found Widespread use in the characterization of biomolecules. ECD produces a radical species that ...

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Section: Resultssupporting

confidence: 92%

Electron-induced dissociation of glycosaminoglycan tetrasaccharides

Wolff

Laremore

Aslam

et al. 2008

J. Am. Soc. Mass Spectrom.

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“…In subsequent studies 48 , RPIP-HPLC-ESI-MS and tandem MS were applied to structurally distinguish isobaric oligosaccharides enzymatically prepared from HA and HN (Fig. 9), having the linkage pattern between glucuronic acid and N -acetylglucosamine residues (in HA, 1→3 and in HN, 1→4) as unique difference.…”

Section: Anticipated Resultsmentioning

confidence: 99%

High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides

Volpi

Linhardt

2010

Nat Protoc

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Glycosaminoglycans (GAGs) have proven to be very difficult to analyze and characterize because of their high negative charge density, polydispersity and sequence heterogeneity. As the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential for developing a structure–activity relationship. Electrospray ionization (ESI) mass spectrometry (MS) is particularly promising for the analysis of oligosaccharides chemically or enzymatically generated by GAGs because of its relatively soft ionization capacity. Furthermore, on-line high-performance liquid chromatography (HPLC)-MS greatly enhances the characterization of complex mixtures of GAG-derived oligosaccharides, providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerization, is presented, together with their HPLC separation, using volatile reversed-phase ion-pairing reagents and on-line ESI-MS structural identification. This analysis provides an oligosaccharide map together with sequence information from a reading frame beginning at the nonreducing end of the GAG chains. The preparation of oligosaccharides can be carried out in 10 h, with subsequent HPLC analysis in 1–2 h and HPLC-MS analysis taking another 2 h.

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“…Fifteen peaks, corresponding primarily to tetrasaccharides, were obtained in the initial semi-preparative HPLC. Chromatographic purification of these peaks were analyzed by LC-MS (Agilent 1100 LC/MSD, Agilent Technologies, Inc., Wilmington, DE) under conditions previously described (20). Generally, 5 g of each sample was loaded through a 5-m Agilent Zorbax SB-C18 (0.5 ϫ 250 mm) column.…”

Section: Preparation Of the Hs Tetrasaccharide Substratesmentioning

confidence: 99%

Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate

Shaya

Zhao

Garron

et al. 2010

Journal of Biological Chemistry

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Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a ␤-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles. Heparin and heparan sulfate (HS)3 glycosaminoglycans (GAGs) are negatively charged, linear polysaccharides composed of repeating disaccharide units of uronic acid and glucosamine residues (GlcN, 2-amino-2-deoxy-␣-D-glucopyranose) (1). Heparin typically contains ϳ90% iduronic acid (IdoA, ␣-Lidopyranosyluronic acid) and 10% glucuronic acid (GlcA, ␤-Dglucopyranosyluronic acid), with a high content of 2-O-sulfo groups on the IdoA residue. The glucosamine residue in heparin is predominantly substituted with N-sulfo groups (GlcNS, where S is sulfo) and 6-O-sulfo groups with a small number of N-acetyl groups and much less frequently with 3-O-sulfo groups. In contrast, HS is somewhat more diverse in its primary structure and characterized by a higher percentage of the GlcA epimer, N-acetyl-substituted GlcN (GlcNAc) and a lower percentage of 2-O-sulfo, 6-O-sulfo, and N-sulfo groups. The modifications in HS are not uniform; rather, they are concentrated within specific regions of the polysaccharide, giving rise to a short, sulfo group containing sequence motifs responsible for the interactions between HS and a diverse repertoire of proteins leading to its multiple biological roles. These complex polysaccharides provide docking sites for numerous protein ligands involved in diverse biological processes, ranging from cancer and angiogenesis, anticoagulation, inflammatory processes, viral and microbial pathogenesis to multiple aspects of development (2-9). Moreover, HS-GAGs are abundant at the cell surface as part of the proteoglycan cell surface receptors (3, 4).Specialized microorganisms express GAG-degrading lyases serving nutritional purposes of both themselves and their vertebrat...

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Tandem MS can distinguish hyaluronic acid from N-acetylheparosan

Cited by 22 publications

References 43 publications

Electron-induced dissociation of glycosaminoglycan tetrasaccharides

Electron-induced dissociation of glycosaminoglycan tetrasaccharides

High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides

Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate

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