The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degr... more The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characte...
The specificity of mild periodate oxidation of 3- and 6-substituted 2-acetamido-2-deoxy-D-galacti... more The specificity of mild periodate oxidation of 3- and 6-substituted 2-acetamido-2-deoxy-D-galactitols and 4- and 6-substituted D-glucitols has been investigated. The products were reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine and the derivatives were analysed by application of liquid secondary-ion mass spectrometry directly to the TLC plates. There was > 95% specificity of cleavage of the C-4-C-5 bond (threo-diol) for the GalNAcol derivatives. The major sites of oxidation for the Glcol derivatives also involved threo-diols. For alpha-Neu5Ac-(2-->6)-GalNAcol, approximately 30% of the products of oxidation involved the sialic acid side chain, and approximately 60% were cleaved at the C-4-C-5 bond of the GalNAcol moiety. The mild periodate oxidation reaction forms part of a strategy for determining the patterns of branching of the cores of O-linked glycoprotein oligosaccharides and other oligosaccharide-alditols.
The sensitivity of detection and extent of information on structure obtainable by liquid-secondar... more The sensitivity of detection and extent of information on structure obtainable by liquid-secondary-ion mass spectrometry (l.s.i.-m.s.) of neoglycolipids on the conventional target probe and directly from the surface of silica plates following t.l.c. has been assessed. Neoglycolipids were derived from malto-oligosaccharides, chitin oligosaccharides, and a range of deoxyhexose-, hexose-, 2-acetamido-2-deoxyhexose-, and sialic acid-containing mammalian oligosaccharides by reductive amination using phosphatidylethanolamine dipalmitoate (PPEADP). Sub-pmol amounts of the maltopentaose-PPEADP derivative applied directly to the target probe provided information on molecular weight, whereas approximately 1 pmol was required when analysed on the silica gel t.l.c. plate. With a biantennary octasaccharide derivative, the sensitivity of detection was 20-50 times lower and the other oligosaccharides had intermediate sensitivities. Information on composition and sequence was obtained readily from fragment ions, using 5 pmol of the maltopentaose derivative and 50 pmol of the octasaccharide derivative on the target probe, and 50 and 200 pmol, respectively, on the silica gel chromatogram. The optimised conditions formed the basis for characterising the structures of the components of mixtures of oligosaccharides generated from glycoproteins.
A charge and size uniform polysaccharide GW2M was extracted with cold water from red alga Gloiope... more A charge and size uniform polysaccharide GW2M was extracted with cold water from red alga Gloiopeltis furcata and purified by strong anion ion-exchange and gel permeation chromatography. Its chemical structure was identified by methylation, (1)H-(1)H COSY, (1)H-(13)C HMQC and (1)H-(13)C HMBC techniques. The experimental data showed that GW2M was composed of galactose (40.3%), 3,6-anhydro-galactose (34.1%) and sulfate (24.8%) with an average molecular mass of 20.6 kDa. The results proved GW2M was a linear repeating sequence of alternating (1→3)-linked 6-O-sulfated-β-d-galactose (G6S) and (1→4)-linked 3,6-anhydro-α-l-galactose (A) which made it to be an ideal 6-O-sulfated-agarose. The sequences of serial oligosaccharides prepared by mild acid and reductive acid hydrolysis from GW2M were confirmed using electrospray collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2012
The aim is to analyze the structure, anticoagulant and antithrombotic activities of a sulfated fu... more The aim is to analyze the structure, anticoagulant and antithrombotic activities of a sulfated fucan isolated from sea cucumber Isostichopus badionotus (fucan-Ib). Fucan-Ib was hydrolyzed under mild acid conditions. The oligosaccharide fragments were fractionated by gel-filtration chromatography and the structures were determined by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation and two-dimensional NMR. Anticoagulant activities were measured by activated partial thromboplastin, thrombin and prothrombin times, and by in vitro inhibition experiments with factors IIa and Xa. Antithrombotic activities were determined in vitro by measuring the length and weight of the thrombus generated. The linear polysaccharide sequence of fucan-Ib was deduced from the structures of its oligosaccharide fragments produced by acid hydrolysis. Under mild conditions, the glycosidic bonds between the non-sulfated and 2,4-O-disulfated fucose residues were selectively cleaved and highly ordered oligosaccharide fragments with a tetrasaccharide repeating unit [→3Fuc(2S,4S)α1→3Fuc(2S)α1→3Fuc(2S)α1→3Fucα1→]n were obtained. In in vitro assays fucan-Ib showed good anticoagulant and antithrombotic activities compared with heparin and the fucosylated chondroitin sulfate isolated from the same source (fCS-Ib). The two polysaccharides, fucan-Ib and fCS-Ib, differ in the mechanism of action; the former exhibited activity mainly by potentiation of antithrombin acted on thrombin and factor Xa whereas the latter mainly through heparin cofactor II. Fucan-Ib has a well defined structure with tetrasaccharide tandem repeats and good anticoagulant and antithrombotic activities. GENERAL IMPORTANCE: Fucan-Ib has a well defined structure and can be readily quality-controlled, and therefore has potential therapeutic value as an affective antithrombotic drug with low risk of bleeding.
Among glycosaminoglycans, polysulfated heparin chains provide the greatest challenge to character... more Among glycosaminoglycans, polysulfated heparin chains provide the greatest challenge to characterization due to high polarity, structural diversity, and sulfate lability. The present report demonstrates how electrospray mass spectrometry (ESMS) can be used to derive compositional information from pure and mixed fractions of heparin tetra- to decasaccharide fragments prepared by controlled digestion of heparin with heparinase I. It also describes an improved procedure for fractionation of heparin oligosaccharides up to octadecasaccharides. Ammonium salts prove to be superior to sodium salts, particularly for analysis of mixed components. In the mass spectrum of a hexasaccharide fraction, the identification of six major mass peaks that represent the known hexasaccharide structures confirms that ESMS analysis of heparin oligosaccharide fragments gives a close representation of their constituent composition. In addition to the previously identified components, several unreported oligosaccharides were detected in the spectra of octa- and decasaccharide fractions. The ESMS identification of the three major species in a decasaccharide fraction was confirmed after HPLC subfractionation and reanalysis. ESMS detection sensitivity of low picomole amounts of oligosaccharides can be readily achieved.
Negative-ion electrospray mass spectrometry (ES-MS) with collision-induced dissociation (CID) and... more Negative-ion electrospray mass spectrometry (ES-MS) with collision-induced dissociation (CID) and MS/MS scanning on a quadrupole-orthoganal time-of-flight instrument provide a sensitive means for structural analysis of neutral underivatized oligosaccharides. Molecular mass information can be readily obtained from the dominant [M - H]- ions in the ES mass spectrum formed with subnanomole amounts of oligosaccharides, and similar sensitivity is available from CID-MS/MS to give structural details. The CID spectra of 14 oligosaccharides demonstrated that sequence and partial linkage information can be derived and isomeric structures can be differentiated. Series of C-type fragment ions give sequence information while the double glycosidic D-type cleavage of a 3-linked GlcNAc or Glc and the saccharide ring fragmentation of the 0,2A-type from 4-linked GlcNAc or Glc can provide partial linkage information. The distinctive D- and A-cleavages are important for differentiation of oligosaccharide type 1 and type 2 chains and to define the blood group H, Le(a), Le(x), Le(b), and Le(y) determinants carried by their fucosylated analogues.
Biochemical and Biophysical Research Communications, 1996
In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin ... more In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin in mouse, glycolipids from tissues and the neutrophilic cell line 32D c13 were tested for E-selectin binding. Kidneys of BALB/c and NMRI mice (but not CBA) and the 32D c13 cells were found to contain minor glycolipid populations that support strongly the binding of murine E-selectin. By chromatogram
The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degr... more The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characte...
The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degr... more The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characte...
The specificity of mild periodate oxidation of 3- and 6-substituted 2-acetamido-2-deoxy-D-galacti... more The specificity of mild periodate oxidation of 3- and 6-substituted 2-acetamido-2-deoxy-D-galactitols and 4- and 6-substituted D-glucitols has been investigated. The products were reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine and the derivatives were analysed by application of liquid secondary-ion mass spectrometry directly to the TLC plates. There was > 95% specificity of cleavage of the C-4-C-5 bond (threo-diol) for the GalNAcol derivatives. The major sites of oxidation for the Glcol derivatives also involved threo-diols. For alpha-Neu5Ac-(2-->6)-GalNAcol, approximately 30% of the products of oxidation involved the sialic acid side chain, and approximately 60% were cleaved at the C-4-C-5 bond of the GalNAcol moiety. The mild periodate oxidation reaction forms part of a strategy for determining the patterns of branching of the cores of O-linked glycoprotein oligosaccharides and other oligosaccharide-alditols.
The sensitivity of detection and extent of information on structure obtainable by liquid-secondar... more The sensitivity of detection and extent of information on structure obtainable by liquid-secondary-ion mass spectrometry (l.s.i.-m.s.) of neoglycolipids on the conventional target probe and directly from the surface of silica plates following t.l.c. has been assessed. Neoglycolipids were derived from malto-oligosaccharides, chitin oligosaccharides, and a range of deoxyhexose-, hexose-, 2-acetamido-2-deoxyhexose-, and sialic acid-containing mammalian oligosaccharides by reductive amination using phosphatidylethanolamine dipalmitoate (PPEADP). Sub-pmol amounts of the maltopentaose-PPEADP derivative applied directly to the target probe provided information on molecular weight, whereas approximately 1 pmol was required when analysed on the silica gel t.l.c. plate. With a biantennary octasaccharide derivative, the sensitivity of detection was 20-50 times lower and the other oligosaccharides had intermediate sensitivities. Information on composition and sequence was obtained readily from fragment ions, using 5 pmol of the maltopentaose derivative and 50 pmol of the octasaccharide derivative on the target probe, and 50 and 200 pmol, respectively, on the silica gel chromatogram. The optimised conditions formed the basis for characterising the structures of the components of mixtures of oligosaccharides generated from glycoproteins.
A charge and size uniform polysaccharide GW2M was extracted with cold water from red alga Gloiope... more A charge and size uniform polysaccharide GW2M was extracted with cold water from red alga Gloiopeltis furcata and purified by strong anion ion-exchange and gel permeation chromatography. Its chemical structure was identified by methylation, (1)H-(1)H COSY, (1)H-(13)C HMQC and (1)H-(13)C HMBC techniques. The experimental data showed that GW2M was composed of galactose (40.3%), 3,6-anhydro-galactose (34.1%) and sulfate (24.8%) with an average molecular mass of 20.6 kDa. The results proved GW2M was a linear repeating sequence of alternating (1→3)-linked 6-O-sulfated-β-d-galactose (G6S) and (1→4)-linked 3,6-anhydro-α-l-galactose (A) which made it to be an ideal 6-O-sulfated-agarose. The sequences of serial oligosaccharides prepared by mild acid and reductive acid hydrolysis from GW2M were confirmed using electrospray collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2012
The aim is to analyze the structure, anticoagulant and antithrombotic activities of a sulfated fu... more The aim is to analyze the structure, anticoagulant and antithrombotic activities of a sulfated fucan isolated from sea cucumber Isostichopus badionotus (fucan-Ib). Fucan-Ib was hydrolyzed under mild acid conditions. The oligosaccharide fragments were fractionated by gel-filtration chromatography and the structures were determined by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation and two-dimensional NMR. Anticoagulant activities were measured by activated partial thromboplastin, thrombin and prothrombin times, and by in vitro inhibition experiments with factors IIa and Xa. Antithrombotic activities were determined in vitro by measuring the length and weight of the thrombus generated. The linear polysaccharide sequence of fucan-Ib was deduced from the structures of its oligosaccharide fragments produced by acid hydrolysis. Under mild conditions, the glycosidic bonds between the non-sulfated and 2,4-O-disulfated fucose residues were selectively cleaved and highly ordered oligosaccharide fragments with a tetrasaccharide repeating unit [→3Fuc(2S,4S)α1→3Fuc(2S)α1→3Fuc(2S)α1→3Fucα1→]n were obtained. In in vitro assays fucan-Ib showed good anticoagulant and antithrombotic activities compared with heparin and the fucosylated chondroitin sulfate isolated from the same source (fCS-Ib). The two polysaccharides, fucan-Ib and fCS-Ib, differ in the mechanism of action; the former exhibited activity mainly by potentiation of antithrombin acted on thrombin and factor Xa whereas the latter mainly through heparin cofactor II. Fucan-Ib has a well defined structure with tetrasaccharide tandem repeats and good anticoagulant and antithrombotic activities. GENERAL IMPORTANCE: Fucan-Ib has a well defined structure and can be readily quality-controlled, and therefore has potential therapeutic value as an affective antithrombotic drug with low risk of bleeding.
Among glycosaminoglycans, polysulfated heparin chains provide the greatest challenge to character... more Among glycosaminoglycans, polysulfated heparin chains provide the greatest challenge to characterization due to high polarity, structural diversity, and sulfate lability. The present report demonstrates how electrospray mass spectrometry (ESMS) can be used to derive compositional information from pure and mixed fractions of heparin tetra- to decasaccharide fragments prepared by controlled digestion of heparin with heparinase I. It also describes an improved procedure for fractionation of heparin oligosaccharides up to octadecasaccharides. Ammonium salts prove to be superior to sodium salts, particularly for analysis of mixed components. In the mass spectrum of a hexasaccharide fraction, the identification of six major mass peaks that represent the known hexasaccharide structures confirms that ESMS analysis of heparin oligosaccharide fragments gives a close representation of their constituent composition. In addition to the previously identified components, several unreported oligosaccharides were detected in the spectra of octa- and decasaccharide fractions. The ESMS identification of the three major species in a decasaccharide fraction was confirmed after HPLC subfractionation and reanalysis. ESMS detection sensitivity of low picomole amounts of oligosaccharides can be readily achieved.
Negative-ion electrospray mass spectrometry (ES-MS) with collision-induced dissociation (CID) and... more Negative-ion electrospray mass spectrometry (ES-MS) with collision-induced dissociation (CID) and MS/MS scanning on a quadrupole-orthoganal time-of-flight instrument provide a sensitive means for structural analysis of neutral underivatized oligosaccharides. Molecular mass information can be readily obtained from the dominant [M - H]- ions in the ES mass spectrum formed with subnanomole amounts of oligosaccharides, and similar sensitivity is available from CID-MS/MS to give structural details. The CID spectra of 14 oligosaccharides demonstrated that sequence and partial linkage information can be derived and isomeric structures can be differentiated. Series of C-type fragment ions give sequence information while the double glycosidic D-type cleavage of a 3-linked GlcNAc or Glc and the saccharide ring fragmentation of the 0,2A-type from 4-linked GlcNAc or Glc can provide partial linkage information. The distinctive D- and A-cleavages are important for differentiation of oligosaccharide type 1 and type 2 chains and to define the blood group H, Le(a), Le(x), Le(b), and Le(y) determinants carried by their fucosylated analogues.
Biochemical and Biophysical Research Communications, 1996
In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin ... more In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin in mouse, glycolipids from tissues and the neutrophilic cell line 32D c13 were tested for E-selectin binding. Kidneys of BALB/c and NMRI mice (but not CBA) and the 32D c13 cells were found to contain minor glycolipid populations that support strongly the binding of murine E-selectin. By chromatogram
The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degr... more The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characte...
Uploads
Papers by Wengang Chai