Carbohydrate Units Research Articles

An easy high-performance liquid chromatography quantification of monosaccharides from heal mucosal tissue glycoproteins in pigs

THE carbohydrate units of glycoproteins modulate significantly the physicochemical and biological properties of the parent proteins. 1 Since the glycosylation machinery 2 depends on the type, as well as on the physiological state, of the organism, tissue, or cell in which the glycoprotein is made, it is now well clearly understood that many pathologic states are characterized by changes in the carbohydrate structure of cellular glycoproteins. Glycosylation changes are therefore becoming widely used as markers for many pathologic situations, including inflammation and cancer. Our interest is focused on small bowel transplantation in pigs in which, despite good control of rejection, the bowel does not seem to work well and a lot of complications prevent good function and long survival. So, the study of the mucosal carbohydrate moieties should help to understand the difficulties associated with an intestinal transplantation. In general, the analysis of the monosaccharide composition of oligosaccharides is performed by several techniques such as gas chromatography (GC), gas chromatographymass spectrometry (GC-MS), 3,4 and nuclear magnetic resonance (NMR). 5 However, there is still a growing demand for easy and sensitive methods of analysis and quantification of samples of biological origin, as the improved sensitive methods recently described in the literature generally concern the compositional analysis of monosaccharides derived from glycoproteins of commercial origin. Here we describe an easy approach to the quantification of the neutral monosaccharides originated from the ileal mucosal glycoprotein content through high-performance liquid chromatography (HPLC).

A General and Highly Efficient Solid Phase Synthesis of Oligosaccharides. Total Synthesis of a Heptasaccharide Phytoalexin Elicitor (HPE)

A new solid phase method for the synthesis of complex oligosaccharides is described. The method involves attachment to phenolic polystyrene of the first carbohydrate unit, through its anomeric position and via a photolabile linker, using dimethylthiomethylsulfonium triflate as an activator. Reiteration of the glycosidation process allows stereocontrolled growth of linear or branched oligosaccharides in high yield. Cleavage from the resin is efficiently achieved photolytically. The application of the method to the total prepn. of the heptasaccharide phytoalexin elicitor (HPE) is described.

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal.

Enzymatic 3-O-sulfation of terminal beta-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Galbeta1,3GalNAc alpha-O-Al, Galbeta1,4GlcNAcbeta-O-Al, Galbeta1,3GlcNAcbeta-O-Al, and mucin-type Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn structures containing a C-3 methyl substituent on either Gal. Two distinct types of Gal: 3-O-sulfotransferases were revealed. One (Group A) was specific for the Galbeta1, 3GalNAc alpha- linkage and the other (Group B) was directed toward the Galbeta1,4GlcNAc branch beta1,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Galbeta-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An examination of activity present in six human sera revealed a specificity of the serum enzyme toward beta1,3 linked Gal, particularly, the T-hapten without beta1,6 branching. Group A was highly active toward T-hapten/acrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galbeta1,3GalNAc alpha-O-Al and Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0-7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereas Mn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography. The following effects upon Gal: 3-O-sulfotransferase activities by fucose, sulfate, and other substituents on the carbohydrate chains were noted. (1) A methyl or GlcNAc substituent on C-6 of GalNAc diminished the ability of Galbeta1,3GalNAc alpha-O-Al to act as an acceptor for Group A. (2) An alpha1,3-fucosyl residue on the beta1,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked beta1,3 to GalNAc alpha-. (3) Lewis x and Lewis a terminals did not serve as acceptors for either Group A or B enzymes. (4) Elimination of Group B activity on Gal in the beta1,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked beta1,3 to GalNAc alpha. (5) Group A activity on Gal linked beta1,3 to GalNAc remained unaffected by 3'-sulfation of the beta1,6 branch. The reverse was true for Group B. (6) The acceptor activity of the T-hapten was increased somewhat upon C-6 sulfation of GalNAc, whereas, C-6 sialylation resulted in an 85% loss of activity. (7) A novel finding was that Galbeta1,4GlcNAcbeta-O-Al and Galbeta1,3GlcNAcbeta-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% inactive and 5- to 7-fold active, respectively, in their ability to serve as acceptors for Group B.

Host-cell-specific glycosylation of HIV-2 envelope glycoprotein.

Neutral complex-type N-glycans of the envelope glycoprotein 120 of HIV-2, propagated in different host cells, display cell-type specific variations. In order to identify typical structural elements, glycans were analysed by gel filtration, by enzymic sequencing and, in part, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The characteristic substituents of di- tri- and tetraantennary carbohydrate units thus observed include N-acetyllactosamine repeats, bisecting N-acetylglucosamine and fucose linked to the chitobiose core as well as to N-acetyllactosamine antennae. Each glycoprotein preparation displayed a characteristic set of glycoforms.

Asparagine-linked carbohydrate chains of inducible rat parotid proline-rich glycoprotein contain terminal beta-linked N-acetylgalactosamine.

Rats treated with daily injection of DL-isoproterenol for 10 consecutive days (25 mg kg(-1) body weight) showed marked induction of a proline-rich glycoprotein (GPRP) of 220 kDa. Proteinase K digestion of GPRP produced a homogeneous glycopeptide with an average chemical composition as follows (residues per mol): Pro4, Glx3, Asx2, Gly1, His1, Thr1, Arg1, GlcNAc5, GalNac1, Man3, Gal2-3, and Fuc1. The structural analysis of the asparagine-linked carbohydrate unit was performed by methylation, periodate oxidation and enzymatic degradation. Methylation studies indicated that the three mannosyl residues were substituted at 1,2-, 1,2,4-, and 1,3,6-positions. Fucose, N-acetylgalactosamine, 1.5 residues of galactose and 0.35 residues of N-acetylglucosamine were terminally located and one galactose residue was 1,4-substituted. Approximately four of the 5 N-acetylglucosamine residues were substituted at 1,4-position and approximately 1 residue of N-acetylglucosamine was substituted at 1,4,6-positions. Periodate oxidation studies and exoglycosidase results were consistent with the methylation data. Based on the results of Smith degradation, methylation and sequential exoglycosidase digestions a triantennary oligosaccharide structure having terminal N-acetylgalactosamine in one of the branches is proposed for the major Asn-linked carbohydrate moiety of GPRP.

Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in beta-linkage to mannose: role in assembly of sialyl-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides.

Rat liver Golgi membranes were found to contain an enzyme that can transfer sulphate from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to C-6 of the terminal GlcNAc in beta-linkage to mannose and has properties indicating that it is involved in the synthesis of the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) sequences observed in the N-linked carbohydrate units of various glycoproteins. Assays performed with [35S]PAPS (Km 0.67 microM) and GlcNAc beta 1-6Man alpha 1-O-Me (GnMaMe) acceptor (Km 0.71 mM) indicated that the sulphotransferase had a pH optimum of approx. 7.0 and is markedly stimulated by Mn2+ ions (maximum approx. 15 mM) and Triton X-100 (0.05-0.1%). Hydrazine/nitrous acid/NaBH4 treatment of the 35S-labelled product yielded radiolabelled 2,5-anhydromannitol(6-SO4). The sulphated GnMaMc product of the GlcNAc-6-O-sulphotransferase could be galactosylated by a rat liver Golgi enzyme that was shown to have the same properties as the UDP-Gal:GlcNAc beta-1,4-galactosyltransferase from bovine milk. Competition studies performed with GlcNAc and GlcNAc-6-SO4 furthermore indicated that the same liver enzyme acted on both acceptors to produce Gal beta 1-4GlcNAc and Gal beta 1-4GlcNAc(6-SO4) with Km values of 1.04 and 1.68 mM respectively. Because the sulphated N-acetyl-lactosaminc could in turn serve as an acceptor for rat liver sialyltransferase, it seems that this enzyme, together with the Golgi galactosyltransferase and the GlcNAc-6-O-sulphotransferase, could act in concert in assembling the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) branches of complex N-linked oligosaccharides.

Silicon-Modified Carbohydrate Surfactants II: Siloxanyl Moieties Containing Branched Structures

Branched siloxanyl-modified carbohydrate surfactants have been synthesized by coupling mono-, di- and poly-functional siloxanes to carbohydrate units either via a branched spacer or by attaching a separate modifying element to a straight-chained structure. Hydrophilic as well as extremely hydrophobic elements have been incorporated successfully. Siloxanyl-modified carbohydrates bearing a secondary amino function were alkylated in regioselective reactions by different epoxides ranging from glycidol- to siloxanyl-modified allyl glycidyl ether derivatives. Alternatively, carbohydrate-modified piperazinyl structures yielded cyclic subunits after alkylation. Structures bearing two identical hydrophilic groups are accessible by alkylation of carbohydrate-modified bisamides. The derivatives synthesized were characterized by means of GC, NMR and elemental analysis.

Silicon‐Modified Carbohydrate Surfactants II: Siloxanyl Moieties Containing Branched Structures

Branched siloxanyl-modified carbohydrate surfactants have been synthesized by coupling mono-, di- and poly-functional siloxanes to carbohydrate units either via a branched spacer or by attaching a separate modifying element to a straight-chained structure. Hydrophilic as well as extremely hydrophobic elements have been incorporated successfully. Siloxanyl-modified carbohydrates bearing a secondary amino function were alkylated in regioselective reactions by different epoxides ranging from glycidol- to siloxanyl-modified allyl glycidyl ether derivatives. Alternatively, carbohydrate-modified piperazinyl structures yielded cyclic subunits after alkylation. Structures bearing two identical hydrophilic groups are accessible by alkylation of carbohydrate-modified bisamides. The derivatives synthesized were characterized by means of GC, NMR and elemental analysis.

Solution structure of a two-base DNA bulge complexed with an enediyne cleaving analog.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 +/- 0.1 angstroms from the pro-S H5' of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.

Definition of the Lectin-like Properties of the Molecular Chaperone, Calreticulin, and Demonstration of Its Copurification with Endomannosidase from Rat Liver Golgi

Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on a Glc alpha 1 --> 3Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreticulin, suggested that this chaperone has lectin-like binding properties. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components. Although optimal binding occurred with Glc1Man9GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to the Glc1Man5GlcNAc stage. The alpha 1 --> 6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc1Man4GlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II.

Site-directed Mutagenesis of Human Lysyl Hydroxylase Expressed in Insect Cells: IDENTIFICATION OF HISTIDINE RESIDUES AND AN ASPARTIC ACID RESIDUE CRITICAL FOR CATALYTIC ACTIVITY

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysyl hydroxylase in insect cells by baculovirus vectors. About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% was soluble at 72 h. Twelve histidines, five aspartates, and all four asparagines that may act as N-glycosylation sites were converted individually to serine, alanine, or glutamine, respectively, and the mutant enzymes were expressed in insect cells. Three histidine mutations and one aspartate mutation appeared to inactivate the enzyme completely. These and other data suggest that histidines 656 and 708 and aspartate 658 provide the three ligands required for the binding of Fe2+ to a catalytic site, whereas the role of the third critical histidine (residue 706) remains to be established. Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme completely, whereas six further histidine mutations and four out of five aspartate mutations had a much more minor effect. Data on the four asparagine mutations suggested that only two of the potential N-glycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity.

Insect cells infected with a recombinant baculovirus express both O- and N-glycosylated forms of the rat glycoprotein hormone alpha-subunit.

Glycoprotein hormones LH, FSH, TSH and chorionic gonadotrophin are heterodimers composed of two non-covalently associated subunits, a common alpha- and a specific beta-subunit. A recombinant baculovirus containing a cDNA encoding the alpha-subunit of rat glycoprotein hormones was constructed. Viral-infected cells expressed, 48 h post infection, 7-10 mg immunoreactive alpha-glycopolypeptide/6 x 10(8) cells, of which 65-6% was able to associate with native LH beta and formed a biologically active heterodimeric hormone that bound to testicular receptors. The treatment with specific glycanases showed that the recombinant alpha-subunit was produced as two differently glycosylated forms; an M(r) 23 000 form which contained exclusively N-linked carbohydrate units and another of M(r) 25 000 which appeared to contain additional 0-linked carbohydrate. Data demonstrated that the alpha-subunit was expressed by insect cells in a manner similar to that by mammalian pituitary gonadotropes producing both the N- and O-glycosylated forms although only the N-glycosylated alpha-subunit is known to be capable of associating with the beta-subunit.

Isolation and partial characterization of a high molecular weight anionic glycoconjugate from transforming growth factor-beta treated bovine articular chondrocyte cultures.

A high molecular weight anionic glycoconjugate was isolated from the media of the transforming growth factor-beta treated chondrocyte cultures by anion-exchange chromatography on DEAE-Sephacel and Mono Q columns and was partially characterized. This high molecular weight anionic glycoconjugate was not detected in the non-treated (control) cultures. Characterization studies showed that the glycoconjugate is a non-reducible, non-collagenous glycoprotein containing O-linked, N-linked, and sialic acid substituted carbohydrate units. The isolated glycoconjugate stained "blue" with Stains All and migrated as a single band on sodium dodecyl sulfate gradient gels (2.5-10% acrylamide - diallyl tartardiamide) at an estimated molecular weight of 540 000. Amino acid and amino sugar analyses showed that it is rich in aspartic acid--asparagine, glutamic acid--glutamine, alanine, proline, and glycine, and contains galactosamine and glucoasmine. This transforming growth factor-beta inducible glycoprotein may be involved in cell differentiation and in the cartilage repair process. It may also be used as a marker to localize the biological activity of transforming growth factor-beta in articular cartilage.

Metathesis Of Ω-Unsaturated Glycosides with Chloro-Aryloxide Complexes of Tungsten, as a New Synthetic Route Leading to Unsaturated Neutral Bolaforms

Abstract Glucosides of unsaturated alcohols, bearing different functional groups on the carbohydrate units, underwent catalytic metathesis reaction affording neutral bolaforms in good yields. Tungsten aryloxo-complexes (1 and 2), due to large steric hindrance, were found to be superior catalysts for this process.

The role of the asparagine-linked oligosaccharides of the alpha-subunit in human thyrotropin bioactivity.

TSH and the gonadotropins (FSH, LH, and hCG) are a family of heterodimeric proteins that share a common alpha-subunit and differ in their hormone-specific beta-subunit. The asparagine-linked (N-linked) oligosaccharides on these hormones are important in signal transduction. The N-linked oligosaccharides on the alpha-subunit have no effect on hCG and hFSH receptor binding, but are critical for their biological activity. Here, we analyzed the role of alpha-subunit N-linked oligosaccharides in human TSH (hTSH) bioactivity by site-directed mutagenesis and gene transfer. This was achieved by mutating the asparagine (Asn) residue in the N-linked glycosylation consensus sequence (Asn-X-Thr/Ser) to aspartic acid. The wild-type hTSH and its variants were expressed in Chinese hamster ovary cells. Wild-type alpha-subunit and its mutants (alpha 1, alpha 2, and alpha(1 + 2)) were efficiently combined with TSH beta-subunit and secreted as dimers. The bioactivity of TSH glycosylation variants was determined by measuring their abilities to stimulate cAMP formation and T3 secretion using a serum-free culture system of human thyroid follicles. Using this system, wild-type hTSH was significantly effective in the stimulation of cAMP formation and T3 secretion. Deletion of the oligosaccharide units from either site 1(alpha 1) or site 2(alpha 2) of the alpha-subunit increased the biological activity of the dimer by about 30%. However, deletion of carbohydrate units from both sites of hTSH alpha-subunit (alpha(1 + 2) resulted in a significant reduction in cAMP formation (by approximately 70%) and T3 secretion (by approximately 40%) compared to that with wild-type hTSH. These findings emphasize the importance of the alpha-subunit N-linked oligosaccharide chains on hTSH bioactivity.

The role of the asparagine-linked oligosaccharides of the alpha-subunit in human thyrotropin bioactivity

TSH and the gonadotropins (FSH, LH, and hCG) are a family of heterodimeric proteins that share a common alpha-subunit and differ in their hormone-specific beta-subunit. The asparagine-linked (N-linked) oligosaccharides on these hormones are important in signal transduction. The N-linked oligosaccharides on the alpha-subunit have no effect on hCG and hFSH receptor binding, but are critical for their biological activity. Here, we analyzed the role of alpha-subunit N-linked oligosaccharides in human TSH (hTSH) bioactivity by site-directed mutagenesis and gene transfer. This was achieved by mutating the asparagine (Asn) residue in the N-linked glycosylation consensus sequence (Asn-X-Thr/Ser) to aspartic acid. The wild-type hTSH and its variants were expressed in Chinese hamster ovary cells. Wild-type alpha-subunit and its mutants (alpha 1, alpha 2, and alpha(1 + 2)) were efficiently combined with TSH beta-subunit and secreted as dimers. The bioactivity of TSH glycosylation variants was determined by measuring their abilities to stimulate cAMP formation and T3 secretion using a serum-free culture system of human thyroid follicles. Using this system, wild-type hTSH was significantly effective in the stimulation of cAMP formation and T3 secretion. Deletion of the oligosaccharide units from either site 1(alpha 1) or site 2(alpha 2) of the alpha-subunit increased the biological activity of the dimer by about 30%. However, deletion of carbohydrate units from both sites of hTSH alpha-subunit (alpha(1 + 2) resulted in a significant reduction in cAMP formation (by approximately 70%) and T3 secretion (by approximately 40%) compared to that with wild-type hTSH. These findings emphasize the importance of the alpha-subunit N-linked oligosaccharide chains on hTSH bioactivity.

Effect of copper co-crystallized with sugar on caries development in desalivated rats.

The purpose of the present study was to explore the effects of varying levels of copper incorporated in sucrose by co-crystallization on caries development in the desalivated rat model and to examine the retention of copper in the oral cavity and its influence on protein and carbohydrate composition of plaque. Forty-eight Sprague-Dawley rats were infected by Streptococcus sobrinus 6715 and desalivated when aged 26 days. Four groups were placed in a König-Höfer programmed feeder and received 17 meals daily at hourly intervals, and essential nutrition (NCP No. 2) by gavage twice daily. The control group (1) received meals of plain sucrose, and groups 2, 3, and 4 received sucrose co-crystallized with 300 ppm Cu2+(2), 150 Cu2+(3), and 75 Cu2+(4). After 3 weeks, the animals were killed, and blood was collected for determination of copper. The lower jaw was removed and sonicated in 0.9% saline solution. Microbial assessment and copper, protein and carbohydrate assays were performed for each animal. Smooth-surface and [sulcal] caries scores were: (1) 92.0[41.4]; (2) 6.1[34.2]; (3) 16.7[32.0]; (4) 24.0[36.0]. Copper in sucrose drastically affected the population of S. sobrinus when compared with the control. Also, Cu2+ sucrose groups had significantly more copper per unit of protein and carbohydrate in jaw suspension than the control group. Concentration of copper in the blood was apparently unaffected by any dietary regimen. It is concluded that Cu2+ in a range of concentrations (75-300 ppm Cu2+) when delivered co-crystallized with sucrose is an effective cariostatic agent. The co-crystallization of a cariostatic agent with sucrose may be an effective method to decrease the cariogenic potential of sucrose.

Structural analysis of the CHO‐derived interleukin‐4 by liquid‐chromatography/electrospray ionization mass spectrometry

AbstractElectrospray (ES) ionization mass spectrometric analysis of CHO‐derived recombinant interleukin‐4 (IL‐4) before and after deglycosylation with peptide: N‐glycosidase F is described. That proved useful not only in deriving the size of the attached carbohydrate components but also in identifying the major glycoforms as the mono‐and disialylated complex‐type N‐linked oligosaccharides. Additional signals arising from glycoforms containing carbohydrate components with more extended or higher branching were also detected. Further mapping of CHO IL‐4 was carried out by combining proteolytic digestion and chromatographic separation of the resulting peptide mixture with on‐line ES mass spectrometric detection. Comparative analysis of the V8 protease digests of CHO IL‐4 and its deglycosylated product revealed the Asn38 N‐glycosylation site. Glycopeptide‐containing fractions were identified by searching the resulting raw ES data for signal pairs whose m/z values differ by the mass of various carbohydrate units adjusted for the signal's charge state (e.g. 97 u difference for a triply charged ES signal of a glycopeptide containing NeuAc units). Furthermore, ES mass spectrometric analysis at elevated orifice potentials allowed the rapid location of the glycopeptides in the total ion current chromatogram by monitoring several carbohydrate‐specific fragment ions. This high orifice‐induced fragmentation is a highly sensitive method for generating sugar diagnostic ions in chromatographically separated components, even when glycopeptide and peptide fragments are co‐eluting.

Primary structure of acidic oligosaccharide-alditols derived from the jelly coat of Ambystoma tigrinum. Occurrence of oligosaccharides with fucosyl(alpha 1-5)[fucosyl(alpha 1-4)]-3-deoxy-D-glycero-D-galacto- nonulosonic acid and fucosyl (alpha 1-2) galactosyl (alpha 1-3)-N- acetylgalactosamine sequences.

Eleven O-glycosidic carbohydrate units derived from the jelly coat of Ambystoma tigrinum were analyzed by 1H-NMR spectroscopy. As previously shown for four other amphibian species, the glycans display a remarkable species specificity. As a characteristic feature, 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) was found difucosylated at C4 and C5, and alpha-Gal attached to C3 of GalNAc-ol. The most representative carbohydrate units are: [formula: see text]

Primary Structure of Acidic Oligosaccharide-alditols Derived from the Jelly Coat of Ambystoma tigrinum. Occurrence of Oligosaccharides with Fucosyl(alpha1-5)[Fucosyl(alpha1-4)]-3-Deoxy-d-Glycero -d-Galacto -Nonulosonic Acid and Fucosyl (alpha1-2) Galactosyl (alpha1-3)-N -Acetylgalactosamine Sequences

Eleven O-glycosidic carbohydrate units derived from the jelly coat of Ambystoma tigrinum were analyzed by 1H-NMR spectroscopy. As previously shown for four other amphibian species, the glycans display a remarkable species specificity. As a characteristic feature, 3-deoxy-d-glycero -d-galacto -nonulo-sonic acid (Kdn) was found difucosylated at C4 and C5, and α-Gal attached to C3 of GalNAc-ol. The most representative carbohydrate units are: