Isotope tag method for quantitative analysis of carbohydrates by liquid chromatography-mass spectrometry

We have previously demonstrated that liquid chromatography/mass spectrometry equipped with a graphitized carbon column (GCC-LC/MS) is useful for the structural analysis of carbohydrates in a glycoprotein. Here, we studied the monosaccharide composition analysis and quantitative oligosaccharide profiling by GCC-LC/MS. Monosaccharides were labeled with 2-aminopyridine and then separated and monitored by GCC-LC/MS in the selective ion mode. The use of tetradeuterium-labeled pyridylamino (d4-PA) monosaccharides as internal standards, which were prepared by the tagging of standard monosaccharides with hexadeuterium-labeled 2-aminopyridine (d6-AP), afforded a good linearity and reproducibility in ESIMS analysis. This method was successfully applied to the monosaccharide composition analysis of model glycoproteins, fetuin, and erythropoietin. For quantitative oligosaccharide profiling, oligosaccharides released from an analyte and a standard glycoprotein were tagged with d0- and d6-AP, respectively, and an equal amount of d0- and d4-PA oligosaccharides were coinjected into GCC-LC/MS. In this procedure, the oligosaccharides that existed in either analyte or a standard glycoprotein appeared as single ions, and the oligosaccharides that existed in both analyte and a standard glycoprotein were detected as paired ions. The relative amount of analyte oligosaccharides could be determined on the basis of the analyte/internal standard ion-pair intensity ratio. The quantitative oligosaccharide profiling enabled us to make a quantitative and qualitative comparison of glycosylation between the analyte and standard glycoproteins. The isotope tag method can be applicable for quality control and comparability assessment of glycoprotein products as well as the analysis of glycan alteration in some diseases.     

The Use of Reverse-Phase Columns for Separation of Unsubstituted Carbohydrates

Reverse-phase chromatography can be used to separate unsubstituted oligosaccharides using water as the eluent. The retention time of the individual oligosaccharides was found to be dependent on the molecular weight of the oligosaccharide and the type and the anomeric configuration of the linkage. This technique can be used for characterizing polysaccharides based on identification of the oligosaccharide fractions obtained by partial acid hydrolysis or for the isolation and purification of oligosaccharides.     

Gas-phase oligosaccharide nonreducing end (GONE) sequencing and structural analysis by reversed phase hplc/mass spectrometry with polarity switching

Here we describe a technique to obtain all the N-linked oligosaccharide structures from a single reversed-phase (RP) HPLC run using on-line tandem MS in both positive and negative ion modes with polarity switching. Oligosaccharides labeled with 2-aminobenzamide (2AB) were used because they generated good ionization efficiency in both ion polarities. In the positive ion mode, protonated oligosaccharide ions lose sugar residues sequentially from the nonreducing end with each round of MS fragmentation, revealing the oligosaccharide sequence from greatly simplified tandem MS spectra. In the negative ion mode, diagnostic ions, including those from cross-ring cleavages, are readily observed in the MS² spectra of deprotonated oligosaccharide ions, providing detailed structural information, such as branch composition and linkage positions. Both positive and negative ion modes can be programmed into the same LC/MS experiment through polarity switching of the MS instrument. The gas-phase oligosaccharide nonreducing end (GONE) sequencing data, in combination with the diagnostic ions generated in negative ion tandem MS, allow both sequence and structural information to be obtained for all eluting species during a single RP-HPLC chromatographic run. This technique generates oligosaccharide analyses at high speed and sensitivity, and reveals structural features that can be difficult to obtain by traditional methods.     

Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis

The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.     

乙酰解方法测定痕量糖链中α1→6联接的分枝点

乙酰解能使糖链中的α1→6联接键优先断离, 本文报道糖链的还原端接上对氨基苯甲酸乙酯(ABEE)后, 应用HPLC分离部分乙酰解的复杂断离产物, 正离子LSIMS测定它们的分子量并判断α1→6联接点。异麦芽八糖α1→6联结序列的测定证实改进后的乙酰解方法能获得满意的结果。此外还测定了μmol IgM样品中寡糖链的两个分枝点。     

Solid-phase synthesis of oligosaccharides and on-resin quantitative monitoring using gated decoupling (13)C NMR

A general strategy for solid-phase oligosaccharide synthesis capable of nondestructive quantitative monitoring has been developed. The synthesis was carried out on TentaGel using thioglycosides as glycosylating agents and dimethylthiomethylsulfonium triflate as the activator. An acylsulfonamide linker was introduced to cleave the oligosaccharide from the resin. The solid-phase reactions were monitored quantitatively by using the inverse gated decoupling technique of (13)C NMR, where two (13)C-enriched markers were used to monitor the reactions: one was (13)C-enriched glycine incorporated as a part of the linker and as an internal standard, and the other was a (13)C-enriched acetyl group used as a protecting group of the glycosylation reagent. A representative synthesis of sialyl Lewis X branched tetrasaccharide was demonstrated.     

Using non-covalent complexes to direct the fragmentation of glycosidic bonds in the gas phase

An investigation of the gas phase chemistry of proton bound oligosaccharide (S)-ligand (L) non-covalent complexes, [S + H + L](+) has been carried out using electrospray ionization (ESI) and tandem mass spectrometry in a quadrupole ion trap. When subjected to collision-induced dissociation (CID), these [S + H + L](+) complexes undergo a range of reactions that can be broadly classified into three main types: (1) Simple dissociation into the individual monomers; (2) cleavage of the oligosaccharide to form B-type sequence ions; (3) cleavage of the ligand species. The second type of reaction is particularly interesting as it can produce a "ladder series" of [B(x) + L](+) ions via ligand induced oligosaccharide bond cleavage. This novel gas phase reaction greatly simplifies the sequencing of oligosaccharides. Both the oligosaccharide and ligand were found to influence the type of reaction pathway observed, with the "ladder series" of [B(x) + L](+) ions being favored for permethylated oligosaccharides and for bifunctional ligands. Cytosine is a particularly good ligand at facilitating the formation of [B(x) + L](+) ions. Analogies with condensed phase chemistry of sugars is made and a potential mechanism for ligand induced oligosaccharide bond cleavage is proposed.     

Oligosaccharide relative quantitation using isotope tagging and normal-phase liquid chromatography/mass spectrometry

The growing interest in the conversion of plant biomass into biofuels has recently highlighted the lack of analytical techniques that are able to profile the fine structures of plant cell-wall polysaccharides. Here we present a new liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) platform called Oligosaccharide Quantitation using Isotope Tagging (OliQuIT) developed for profiling the oligosaccharides derived from glycosyl hydrolase digestion of polysaccharides. The method is demonstrated using different arabinoxylan-derived oligosaccharide samples, which are reductively aminated with either the light (12C6) or heavy (13C6) form of aniline. The complex oligosaccharide mixtures are analysed by capillary normal-phase (NP)-LC and ESI-MS. Importantly, arabinoxylan oligosaccharide isomers are separated by NP-LC and their relative abundance in different samples can be determined from the intensities of ions labeled with the different isotopes. OliQuIT will be of use in multiple applications, including screening for plant varieties with improved saccharification properties, characterizing glycosyl hydrolase specificities and analysing plant glycosyl transferase mutants.     

Determination of edible bird's nest and its products by gas chromatography

A specific gas chromatographic (GC) detection method for edible bird's nest (EBN) based on identifying the composition of the oligosaccharide chain combined with glycoprotein in EBN is developed. Five monoses (D-mannitose, D-galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and N-acetyl neuraminate) that constitute the oligosaccharide chain are detected using GC and GC-mass spectrometry techniques; their characteristic GC spectrum can reliably be regarded as EBN's fingerprint. The peak-area ratios in GC spectrum of those five monoses are found to be fixed; therefore, the GC technique developed in this work can conveniently be used to determine various raw EBNs and their products both qualitatively and quantitatively, distinguishing between fake and genuine EBN rapidly.     

A glycomics platform for the analysis of permethylated oligosaccharide alditols

This communication reports the development of an LC/MS platform for the analysis of permethylated oligosaccharide alditols that, for the first time, demonstrates routine online oligosaccharide isomer separation of these compounds before introduction into the mass spectrometer. The method leverages a high-resolution liquid chromatography system with the superior fragmentation pattern characteristics of permethylated oligosaccharide alditols that are dissociated under low-energy collision conditions using quadrupole orthogonal time-of-flight (QoTOF) instrumentation and up to pseudo MS(3) mass spectrometry. Glycoforms, including isomers, are readily identified and their structures assigned. The isomer-specific spectra include highly informative cross-ring and elimination fragments, branch position specific signatures, and glycosidic bond fragments, thus facilitating linkage, branch, and sequence assignment. The method is sensitive and can be applied using as little as 40 fmol of derivatized oligosaccharide. Because permethylation renders oligosaccharides nearly chemically equivalent in the mass spectrometer, the method is semiquantitative and, in this regard, is comparable to methods reported using high field NMR and capillary electrophoresis. In this postgenomic age, the importance of glycosylation in biological processes has become clear. The nature of many of the important questions in glycomics is such that sample material is often extremely limited, thus necessitating the development of highly sensitive methods for rigorous structural assignment of the oligosaccharides in complex mixtures. The glycomics platform presented here fulfills these criteria and should lead to more facile glycomics analyses.     

Preparation of tagged glucose as a key intermediate in the synthesis of branched oligosaccharides

Tagged and activated d-glucose was introduced as a building block for branched oligosaccharides. This building block was the oligosaccharide branching point at which selectively the C-2 followed by the C-3 position can be glycosylated. After the activation of the C-1 position by oxidation of a thiophenyl group, the newly formed tagged oligosaccharide can be used as a glycoside donor. Synthetic procedures for the preparation of phthalimide-based tag molecules as well as tagged monosaccharides are presented.     

Combinatorial synthesis of an oligosaccharide library by using beta-bromoglycoside-mediated iterative glycosylation of selenoglycosides: rapid expansion of molecular diversity with simple building blocks

A new method for constructing an oligosaccharide library composed of structurally defined oligosaccharides is presented based on an iterative glycosylation of selenoglycosides. Treatment of 2-acyl-protected selenoglycosides with bromine selectively generates beta-bromoglycosides, which serve as glycosyl cation equivalents in the oligosaccharide synthesis. Thus, the coupling of the bromoglycosides with another selenoglycoside affords the corresponding glycosylated selenoglycosides, which can be directly used to next glycosylation. The iteration of this sequence allows the synthesis of a variety of oligosaccharides including an elicitor active heptasaccharide. A characteristic feature of the iterative glycosylation is that glycosyl donors and acceptors with the same anomeric reactivity can be selectively coupled by activation of the glycosyl donor prior to coupling with the glycosyl acceptor. Therefore, same selenoglycosides can be used for both the glycosyl donors and the acceptors. This feature has been exemplified by a construction of an oligosaccharide library directed to elicitor-active oligosaccharides. The library composed of stereochemically defined oligoglucosides with considerable structural diversity can be constructed starting from simple selenoglycosides.     

Rapid synthesis of oligosaccharide moieties of globotriaosylceramide using fluorous protective group

The use of the Bfp (bisfluorous chain type propanoyl) group as a fluorous protective group made it possible to rapidly synthesize galabiose and the Gb3 oligosaccharide derivatives by a simple fluorous-organic extraction purification. The fluorous oligosaccharide synthesis using the Bfp group is an excellent strategic alternative to solid phase oligosaccharide synthesis, and removes some of the disadvantages of the solid phase method.     

Superbeads: immobilization in "sweet" chemistry

Enzymatic oligosaccharide synthesis using recombinant glycosyltransferases is able to overcome the difficulties associated with chemical methods. Nonetheless, sugar nucleotide regeneration cycles are necessary for the glycosylation. The multistep enzyme reaction can be efficiently carried out on superbeads that are prepared by immobilizing multienzyme mixtures on bead support through fused binding domains.     

Syntheses,Conformations and X-Ray Structure Analyses of the Saccharide Chains from the Core Regions of Glycoproteins

The covalently bound carbohydrate moiety in glycoproteins can stabilize the protein molecule intramolecularly, or it may have an intermolecular function as receptor in biological recognition. The discovery of these biological phenomena has led to a renaissance of the chemistry and biochemistry of carbohydrates. Both N-glycoproteins as well as O-glycoproteins contain special, invariant oligosaccharide chains in the protein-binding region, which occur again in all glycoproteins, and are described as the “core regions.” This review describes the various methods of oligosaccharide synthesis that may be used to arrive at the basic core structures by chemical means. Methods of oligosaccharide synthesis have improved so much that it is possible to synthesize complex lactosamine-type structures, and “bisected”-type structures up to nona- and undecasaccharides respectively. Oligosaccharide chains are considerably less flexible than peptide chains. Using modern methods of NMR spectroscopy, their preferred solution conformation can readily be determined. In the case of one branched octasaccharide, a comparison of the conformations in solution and in the crystal is possible. Oligosaccharides may be linked to the amide group of an asparagine, or to the hydroxyl groups of serine or threonine. By using suitable protecting groups, the glycosyl amino acids obtained can be extended with further amino acids at the N- or C-terminus, thus arriving at the desired glycopeptide sequences. In the linkage region, glycopeptides prefer certain conformations. Future research into glycoprotein functions may involve the synthesis and biochemical study of modified glycoprotein segments.     

Synthesis of differentially protected ribitol derivatives from 3,4-O-benzylidene-d-ribono-1,5-lactone

Several differentially protected ribitol derivatives were synthesised using 3,4-O-benzylidene-d-ribono-1,5-lactone as versatile starting compounds for oligosaccharide synthesis. The obtained ribitol derivatives allow the regiospecific coupling of glycosyl donors to either of the hydroxyl groups of ribitol and can be applied for the preparation of polyhydroxylated compounds.     

Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography.

A gel permeation chromatographic method for quantifying neutral oligosaccharide fractions from human milk has been developed. Oligosaccharides from monofucosyllactoses to trifucosyllacto-N-hexaoses were separated according to size on a Fractogel TSK HW 40 (S) column. Refractive index detection of monofucosyllactoses to difucosyllacto-N-tetraoses yielded a constant mass response factor of ca. 1 relative to glucose. After the addition of glucose as an internal standard, oligosaccharides were isolated from human milk by ethanol precipitation or two ultrafiltration procedures. The oligosaccharide concentrations found by the ultrafiltration procedures were significantly lower (significance level 0.05) than those determined by the ethanol precipitation procedure.     

One-pot chemo-, regio-, and stereoselective double-differential glycosidation mediated by lanthanide triflates

Nuanced activation of n-pentenyl, thioglycoside, and trichloroacetimidate donors by lanthanide salts coupled with donor/acceptor matching can simplify oligosaccharide assembly. Thus, a one-pot, double-differential glycosidation process can be designed, in which an n-pentenyl acceptor-diol is chemo- and regioselectively glycosidated by using an n-pentenyl ortho ester under the agency of Yb(OTf)(3)/NIS followed by in situ addition of a 2-O-acylated trichloroacetimidate or ethyl thioglycoside to effect stereoselective glycosidation at the remaining OH.     

S-benzoxazolyl as a stable protecting moiety and a potent anomeric leaving group in oligosaccharide synthesis

As a part of a program for developing new versatile building blocks for stereoselective glycosylation and convergent oligosaccharide synthesis, we demonstrated that S-benzoxazolyl (SBox) glycosides are stable toward major protecting group manipulations employed in carbohydrate chemistry. On the other hand, they can be glycosidated under relatively mild reaction conditions to afford either 1,2-trans or 1,2-cis-linked disaccharides. Selective and chemoselective activations of the SBox moiety were also proved to be feasible, which was demonstrated by synthesizing a number of oligosaccharide sequences.     

Unusual covalent bond-breaking reactions of beta-cyclodextrin inclusion complexes of nucleobases/nucleosides and related guest molecules

Host-guest complexes between nucleobases or nucleosides and beta-cyclodextrin can be observed by electrospray ionization mass spectrometry (ESI-MS) and their relative abundances appear to correlate with the condensed-phase binding order. Using Fourier transform ion cyclotron resonance mass spectrometry, the extent of the interactions between the host oligosaccharide and guest species have also been examined for permethylated beta-cyclodextrin : adenine/deoxyadenosine and permethylated maltoheptaose : adenine/deoxyadenosine using gas-phase exchange reactions with the gaseous amines, n-propylamine and ethylenediamine. The ease of guest exchange in the gas-phase follows the order : deoxyadenosine > adenine > deoxycytidine > cytosine, which is in contrast to their relative binding order in solution. Collision-induced dissociation (CID) has been used to probe the fragmentation behavior of oligosaccharide : nucleobase/nucleoside complexes. Under these conditions the inclusion complexes either (a) dissociate, (b) result in cleavage of the host oligosaccharide or (c) result in cleavage of the guest molecule. This study has shown that the preferred dissociation pathway of these complexes depends on the structures of both the cyclodextrin and guest molecule.