Fluorescent staining of glycoproteins on polyvinylidene difluoride membrane with 8-aminonaphthalene-1,3,6-trisulfonate.

Here, we describe a simple and sensitive method that allows fluorescent detection of glycoproteins on polyvinylidene difluoride (PVDF) membrane. We used periodic acid oxidation of carbohydrate chains of glycoproteins and fluorescent labeling with 8-aminonaphthalene-1,3,6-trisulfonate (AN-TS) by reductive amination. We developed an additional method to enhance the ability of PVDF to absorb glycoproteins by using non-glycoprotein lectin, such as wheat germ agglutinin (WGA), as a link between the PVDF membrane and glycoproteins, resulting in considerably increased detection sensitivity to glycoproteins.     

The role of N-linked oligosaccharides in glycoprotein function.

N-linked glycoproteins include such biologically important molecules as cell-surface receptors, cell-adhesion molecules, immunoglobulins and other serum proteins, and tumor antigens. Investigating the role of carbohydrate in glycoprotein function has included the use of glycosylation inhibitors or site-directed mutagenesis of specific glycosylation sites to prevent the addition of carbohydrate, or glycosylation processing inhibitors or animal cell glycosylation mutants to alter carbohydrate structure. In some proteins, glycosylation plays an important role in recognition, while in others, it may stabilize and/or control the conformation of the protein. The cloning of genes in bacteria or lower eukaryotes--with the goal of producing biologically active proteins for biotechnological purposes--necessitates a better understanding of the role of specific carbohydrate structures.     

The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device.

Various monosaccharides, oligosaccharides and small polysaccharides were labelled covalently at their reducing end groups with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS), and the resulting fluorescent derivatives were separated by high-resolution PAGE. The electrophoretic mobilities of the labelled saccharides are related largely to the compounds' Mr values, but they are also influenced by the individual chemical structures of the saccharides. Various positional isomers and some epimers, for instance galactose and glucose, were resolved. Oligosaccharide and small polysaccharide derivatives, prepared from an enzymic digest of starch, each differing in size by a single hexose residue and with a range of degrees of polymerization from 2 to 26, were all resolved in a single gel. The method was relatively rapid and simple to perform. It enabled multiple samples to be analysed in parallel with high sensitivity. The fluorescent-labelling procedure was virtually quantitative. As little as 1 pmol of ANTS-labelled saccharide was detected photographically when the gels were illuminated by u.v. light. When the gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrate moieties of glycoconjugates and other naturally occurring oligosaccharides.     

Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein

Newcastle disease virus sialidase was found to exhibit strict specificity for hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage contained in glycoprotein oligosaccharides both N-linked to asparagine and O-linked to threonine or serine under conditions that left oligosaccharides containing the NeuAc alpha 2 leads to 2 leads to 6Gal and NeuAc alpha 2 leads to 6GallNAc linkages intact. This was determined, in part, by examining the viral sialidase for its ability to hydrolyze glycoprotein oligosaccharides derivatized with purified sialyltransferases to contain the [14C]NeuAc alpha 2 leads to 3Gal, [14C]NeuAc alpha 2 leads to 6GalNAc, and [14C]NeuAc alpha 2 leads to 6Gal linkages. The viral sialidase was also tested for hydrolysis of the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages on the N-linked oligosaccharides of alpha 1-acid glycoprotein. Selective hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage was shown by periodate oxidation and by 500-MHz 1H-NMR spectroscopy of native and sialidase-treated glycopeptides. The NMR spectra, together with composition data, further indicated that the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages were localized to specific branches of the major tri- and tetraantennary oligosaccharides of alpha 1-acid glycoprotein. The results indicate that the Newcastle disease virus sialidase can initiate the selective degradation of N-linked oligosaccharide branches containing the NeuAc alpha 2 leads to 3Gal linkage.     

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site.     

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).     

毛细管电泳分离寡糖衍生物及其电泳行为研究

将葡聚糖部分酸水解成寡糖混合物,经ＡＮＴＳ胺化还原衍生,在ｐＨ２．５、５０ｍｍｏｌ／Ｌ磷酸缓冲液（含或不含１０ｍｍｏｌ／Ｌ ＴＥＡ）中,以及在ｐＨ９．３、１００ｍｍｏｌ／Ｌ硼砂缓冲液中用毛细管电泳分离衍生物,分别得到１至２１和１至１８个聚合度的衍生物电泳梯度图谱。作出了准确定位的毛细管电泳双向电泳图。对电泳行为研究发现,在低ｐＨ磷酸电泳缓冲液中,衍生物相对电迁移率（μｅｐ）ｒ与（Ｍｒ＾－２／３）、     

Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides

The variant surface glycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysis of the intact protein and of glycopeptides. Processing has been examined by comparing glycan structures purified from an immature intracellular form (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAc2) at Asn-432 in both the immature 58-kDa and mature 59-kDa forms. In contrast, the "core" oligosaccharide of Asn-419 (Man3-GlcNAc2) appears to be nearly quantitatively processed to a complex biantennary structure [Gal-GlcNAc-Man)2-Man-GlcNAc2) during VSG maturation. The asparagine-linked structures at Asn-419, but not those at Asn-432, are resistant to endo-beta-N-acetylglucosaminidase H within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This report provides a direct demonstration of the processing of such a glycolipid anchor following its attachment to protein.     

Selective fluorescence detection of 8-oxoguanosine with 8-oxoG-clamp

8-oxoguanosine, which is derived from the oxidation of guanosine (dG), is known to induce transversion mutations (G:C-->T:A) in DNA. The compounds with a small molecular weight for recognizing 8-oxoG were designed on the basis of the structure of the G-clamp, which is reported to have selective affinity toward guanosine. The G-clamp derivatives with the additional binding units toward 8-oxoG were effectively synthesized and named "8-oxoG-clamps." The 8-oxoG-clamp completely discriminated 8-oxoG from other nucleosides by fluorescence quenching.     

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.     

Rapid and simple preparation of N-linked oligosaccharides by cellulose-column chromatography.

As a means of preparing N-linked oligosaccharides from hydrazinolysates of glycoproteins in a rapid and simple manner, a method has been developed using cellulose-column chromatography. Hydrazinolysates of human IgG, containing a series of biantennary complex type oligosaccharides, were applied to a cellulose column equilibrated with (4:1:1, v/v) 1-butanol-ethanol-water. The N-linked oligosaccharides were eluted with (1:1, v/v) ethanol-water, and analyzed by HPLC in combination with sequential glycosidase digestion. The oligosaccharides, with or without sialic acid, were quantitatively recovered in the fraction eluted with (1:1, v/v) ethanol-water without UV-detectable contamination by impurities derived from protein or the cellulose. Other types of N-linked oligosaccharides of alpha1-acid glycoprotein (tetraantennary complex-type), ovalbumin (hybrid-type), and ribonuclease B (high mannose-type) were also quantitatively prepared from the hydrazinolysates by elution of the cellulose column with (1:1, v/v) ethanol-water and these had as high a quality as those prepared by conventional paper chromatography.     

Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells

To analyze the role of glucose trimming and reglucosylation in the binding of substrate proteins to calnexin in the endoplasmic reticulum (ER) of living cells, we made use of the thermosensitive vesicular stomatitis virus tsO45 glycoprotein (G protein). At nonpermissive temperature the G protein failed to fold completely and remained bound to calnexin. When the cells were shifted to permissive temperature, complete folding occurred accompanied by glucosidase-mediated elimination of calnexin-G protein complexes. If release from calnexin was blocked during the temperature shift by inhibiting the glucosidases, folding occurred, albeit at a reduced rate. In contrast, when unfolded by a shift from permissive to nonpermissive temperature, the G protein was reglucosylated rapidly and became capable of rebinding to calnexin. The rate at which calnexin binding occurred showed a 20-min delay that was explained by accumulation of the G protein in calnexin-free exit sites of the ER. These contained the glucosyltransferase responsible for reglucosylation of misfolded glycoproteins but had little or no calnexin. After unfolding and reglucosylation, the G proteins moved slowly from these structures back to the ER where they reassociated with the chaperone. Taken together, these results in live cells fully supported the lectin-only model of calnexin function. The ER exit sites emerged as a potentially important location for components of the quality control system.     

The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common alpha and a hormone specific beta subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the alpha and beta subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (alpha, FSHbeta and hCGbeta) asparagine-linked (N-linked) oligosaccharides. CGbeta subunit is distinguished among the beta subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure-function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

N-Acetylneuraminic acid and N-glycolylneuraminic acid in the O-linked oligosaccharides of a tumor cell glycoprotein. Incorporation and distribution

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, which differ in several cell surface properties, contain a major mucin-type glycoprotein, termed ASGP-1. The sialic acid content of MAT-C1 ASGP-1 is 2-3-fold greater than MAT-B1 ASGP-1 (Sherblom, A. P., Buck, R. L., and Carraway, K. L. (1980) J. Biol. Chem. 255, 783-790). Sialic acid analysis demonstrated that, whereas MAT-C1 ASGP-1 contained approximately equal amounts of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl), MAT-B1 ASGP-1 was devoid of NeuGl. MAT-B1 microsomes also did not contain NeuGl. MAT-B1 cells incubated with [3H]N-acetylmannosamine did not synthesize either labeled CMP-NeuGl or free NeuGl, even though the CMP-sialic acid synthetase was active with the substrate NeuGl. Thus, MAT-B1 cells may be deficient in the enzyme N-acetylneuraminate monooxygenase. The O-linked oligosaccharides from both MAT-B1 and MAT-C1 ASGP-1 have been shown to contain a core tetrasaccharide Gal(beta 1-4)GlcNAc(beta 1-6)(Gal(beta 1-3]GalNAc in which both galactose residues may be linked to additional sugars (Hull, S. R., Laine, R. A., Kaizu, T., Rodriquez, I., and Carraway, K. L. (1984) J. Biol. Chem. 259, 4866-4877). The distribution of NeuAc and NeuGl between the two galactose termini of the core tetrasaccharide was examined for MAT-C1 ASGP-1. Oligosaccharides were released by alkaline-borohydride treatment of MAT-C1 ASGP-1 which had been labeled with [14C]glucosamine and galactose oxidase/B3H4. Following fractionation by Bio-Gel P-4, DEAE-Sephadex, and high-performance liquid chromatography, oligosaccharides were analyzed for NeuAc and NeuGl and for susceptibility to digestion with beta-galactosidase. Three disialylated oligosaccharides were identified containing 2 mol of NeuAc (5.5% recovery), 2 mol of NeuGl (4.5%), or 1 mol each of NeuAc and NeuGl (11.1%). For monosialylated oligosaccharides, NeuGl appeared preferentially associated with the Gal(beta 1-4)GlcNAc terminus (9.0%), whereas significant amounts of oligosaccharide containing NeuAc at both the Gal(beta 1-3)GalNAc (2.6%) and Gal(beta 1-4)GlcNAc (4.5%) termini were detected. Each of the major qualitative differences between MAT-B1 and MAT-C1 oligosaccharides, including the presence of NeuGl (MAT-C1), sulfate (MAT-B1), and alpha-linked galactose (MAT-B1), occurs at the Gal(beta 1-4)GlcNAc terminus.     

High-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry for analysis of oligosaccharides derivatized by reductive amination and N,N-dimethylation

Milk oligosaccharides derivatized by reductive amination with benzylamine followed by N,N-dimethylation (DMBA-oligosaccharides), were analyzed by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry (HPLC/ESI-ITMS). Separation of DMBA-oligosaccharides was achieved on a graphitized carbon column eluted with aqueous acetonitrile and the DMBA-oligosaccharides were detected by positive-ion mode ESI-ITMS allowing sample amounts down to approximately 30fmol of single DMBA-oligosaccharides injected on the HPLC column. MS/MS operation of the mass spectrometer resulted in the detection of diagnostic fragments, mainly belonging to the Y-series, allowing differentiation between isomeric milk oligosaccharides. HPLC/ESI-ITMS/MS/MS experiments indicated the migration of fucose residues of the DMBA milk oligosaccharides to the modified reducing end glucose residue during analysis, a migration previously only observed for proton adduct ions.     

Primary structure determination of seven novel N-linked carbohydrate chains derived from hemocyanin of Lymnaea stagnalis. 3-O-methyl-D-galactose and N-acetyl-D-galactosamine as constituents of xylose-containing N-linked oligosaccharides in an animal glycoprotein

Hemocyanin from the freshwater snail Lymnaea stagnalis is a high-molecular-mass copper-containing glyco-protein which functions as oxygen carrier in the hemolymph. To release the carbohydrate chains, the protein was digested by pronase followed by hydrazinolysis and reduction. The oligosaccharide-alditols were purified by gel permeation chromatography on Bio-Gel P-4, followed by HPLC on a Lichrosorb-NH2 column. Using 500-MHz 1H-NMR spectroscopy, in conjunction with sugar, methylation and deamination analysis, the following seven novel primary oligosaccharide structures could be unravelled. (Formula: see text).     

Sulfated N-linked oligosaccharides in mammalian cells. I. Complex-type chains with sialic acids and O-sulfate esters

The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines (Freeze, H. H., and Varki, A. (1986) Biochem. Biophys. Res. Commun. 140, 967-973). Here we report improved methods to metabolically label cell lines with 35SO4 and to release sulfated N-linked oligosaccharides with peptide:N-glycosidase F as well as the partial structure of some of these novel oligosaccharides. The released 35SO4-labeled chains from Chinese hamster ovary (CHO) cells and bovine pulmonary artery endothelial cells (CPAE) were characterized by gel filtration, anion exchange and lectin affinity chromatography, and various enzymatic and chemical treatments. Each cell line contains a class of sulfated oligosaccharide chains bearing from two to six negative charges in varying combinations of O-sulfate esters and sialic acids. These molecules represent a significant proportion of both the total 35SO4 label and the total anionic N-linked oligosaccharides. They are also relatively enriched in a CHO mutant that is deficient in glycosaminoglycan chain synthesis. Lectin affinity chromatography of such molecules from CPAE cells indicates that the majority are sialylated multiantennary complex-type chains. The sulfate esters are exclusively of the primary type. Sequential exoglycosidase digestions, including beta-hexosaminidase A treatment at low pH, demonstrate that at least one-third of these sulfate esters are found in the following structure, (formula; see text) where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., Roux, L., Varki, A., and Freeze, H. H. (1988) J. Biol. Chem. 263, 8890-8896).     

Primary structure of the low-molecular-weight carbohydrate chains of Helix pomatia alpha-hemocyanin. Xylose as a constituent of N-linked oligosaccharides in an animal glycoprotein

alpha-Hemocyanin of Helix pomatia is a copper-containing glycoprotein which serves as an oxygen carrier in the hemolymph. Its carbohydrate moiety has as constituents fucose, xylose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine, and N-acetyl-glucosamine residues. Alkaline borhydride did not split off any carbohydrate material, suggesting the absence of O-glycosidic chains. The N-glycosidic carbohydrate chains of this glycoprotein were liberated by hydrazinolysis of a Pronase digest then fractionated as alditols on Bio-Gel P-4. The fractions containing the low-molecular-weight glycans were investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar and methylation analysis. The largest, and most abundant, compound was established to be: (Formula: see text). Another compound was characterized as the afuco analogue of this structure. H. pomatia alpha-hemocyanin is the first example of an animal glycoprotein having xylose as a constituent of N-glycosidic carbohydrate chains.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.