Structural Analysis Of Oligosaccharides Research Articles

Structure characterization of a highly branched galactan from the slug Vaginulus alte and its utilization by human gut microbiota

The slug Vaginulus alte is used as folk medicine in China, but the structure and activities of its galactan components remain to be clarified. Here, the galactan from V. alte (VAG) was purified. The Mw of VAG was determined as ~28.8 kDa. Chemical composition analysis showed that VAG was composed of d-galactose (75 %) and l-galactose (25 %). To elucidate its precise structure, a series of disaccharides and trisaccharides were purified from mild acid hydrolyzed VAG and their structures were characterized by 1D/2D NMR spectroscopy. Based on methylation analysis and structural analysis of oligosaccharides, VAG was elucidated as a highly branched polysaccharide and mainly composed of (1 → 6)- or (1 → 3)-linked β-d-galactose, and distinct (1 → 2)-linked α-l-galactose. The investigation of probiotic effects in vitro revealed that VAG could promote the growth of B. thetaiotaomicron and B. ovatus, while had no effect on the growth of L. acidophilus, L. rhamnosus, B. longum subsp. infantis and B. animalis subsp. lactis, but dVAG-3 with Mw ~1.0 kDa could promote the growth of L. acidophilus. These results will provide insights into specific structures and functions of polysaccharides from the V. alte.

Oligosaccharides from Traditional Chinese Herbal Medicines: A Review of Chemical Diversity and Biological Activities.

Most of traditional Chinese herbal medicine (TCHM) substances come from medicinal plants, among which oligosaccharides have gradually attracted widespread attention at home and abroad due to their important biological activities and great medicinal potential. Numerous in vitro and in vivo experiments exhibited that oligosaccharides possess various activities, such as antitumor, anti-oxidation, modulate the gut microflora, anti-inflammatory, anti-infection, and immune-regulatory activities. Generally, biological activities are closely related to chemical structures, including molecular weight, monosaccharide composition, glycosidic bond connection, etc. The structural analysis of oligosaccharides is an important basis for studying their structure-activity relationship, but the structural diversity and complexity of carbohydrate compounds limit the study of oligosaccharides activities. Understanding the structures and biological functions of oligosaccharides is important for the development of new bioactive substances with natural oligosaccharides. This review provides a systematic introduction of the current knowledge of the chemical structures and biological activities of oligosaccharides. Most importantly, the reported chemical characteristics and biological activities of the famous TCHM oligosaccharides were briefly summarized, including Morinda officinalis, Rehmannia glutinosa, Arctium lappa, Polygala tenuifolia, Panax ginseng, Lycium barbarum and Astragalus membranaceus. TCHM oligosaccharides play an important role in nutrition, health care, disease diagnosis and prevention as well as have broad application prospects in the field of medicine.

Structure and composition of blueberry fiber pectin and xyloglucan that bind anthocyanins during fruit puree processing

Blueberry is a well-known source of antioxidants and prebiotic dietary fiber yet the fate of these health-beneficial ingredients during fruit processing it is not understood. Detailed composition and structure of blueberry pomace (PF) remaining after commercial blueberry fruit puree processing, as well as the water soluble and water insoluble fractions, were determined. The PF was comprised of dietary fiber (60.8%) that was largely insoluble (46.2% vs. 14.6% soluble dietary fiber) pectin, xyloglucan, arabinoxylan and mannan polysaccharides, fructose (11.22%) and glucose (10.37%) free sugars, protein (9%), fat (5%), and anthocyanins (6444.5 μg/g). The most abundant anthocyanins detected in PF were malvidin 3-arabinoside and peonidin 3-glucoside and the pectin was determined to be methyl-esterified and acetylated rhamnogalacturonan I with a 4,5-unsaturated function at the non-reducing end based on oligosaccharide structural analysis. Blueberry PF xyloglucan oligosaccharide side chain structures included xylose, arabinose and galactose consistent with arabino-xyloglucan structure. Microwave-assisted extraction of blueberry PF (pH 2 for 10 min at 80 °C) was performed to more clearly elucidate pectin structure, and revealed a pectin polymer with high molar mass (1072 kDa) and viscosity that was dependent upon the molar mass. A random coil shape was confirmed for both the blueberry PF and microwave extracted blueberry pectin, which agreed with previous reports that flexible blueberry pectin contributes to strong anthocyanin binding. In summary, the blueberry pomace anthocyanin content was slightly higher than that reported for fresh whole blueberry fruit, which suggests that most blueberry anthocyanins adhered to the pomace fiber, and most likely the insoluble pectin, during and after processing.

A regular fucan sulfate from Stichopus herrmanni and its peroxide depolymerization: Structure and anticoagulant activity

Marine sulfated polysaccharides have aroused widespread concern for their various structures and bioactivities. Peroxide depolymerization is a common strategy in analysis of structures and structure-activity relationships of polysaccharides. However, confirming the depolymerization process and exact structures of the degradation products is still a considerable challenge. This study reported the structures of a fucan sulfate (FS) from sea cucumber Stichopus herrmanni and its depolymerized products (dFS) prepared by peroxide degradation. The FS was elucidated with a highly regular structure, {-3)-L-Fuc2S-(α1-}n. Structure analysis of oligosaccharides purified from dFS suggested that peroxide degradation involved in cleavage of glycosidic bonds and oxidative modification of reducing end of sugar residue, while no break in sugar ring was observed. Both FS and series of dFSs exhibited significant anticoagulant activities due to their anti-thrombin effects in presence of heparin cofactor II and their potencies were related to their molecular sizes, dFS with ∼ 20 kDa showed the strongest activity.

Urine oligosaccharide tests for the diagnosis of oligosaccharidoses

AbstractThis review discusses the development of capillary electrophoresis with laser-induced detection and mass spectrometry techniques for the analysis of urinary oligosaccharides to screen for human oligosaccharidoses and related disorders. Capillary electrophoresis is suitable for the analysis of urinary oligosaccharides. It has a high resolution efficiency, and when it is coupled to a laser-induced fluorescence detector system, it offers an optimal analytical sensitivity. Mass spectrometry techniques have evolved as powerful tools for glycan analyses, are important tools for the analysis of oligosaccharide structures, and offer precise results, analytical versatility, very high sensitivity, high precision and high speed. Mass spectrometry is tolerant of mixtures and is a natural choice for the analysis of this class of molecules. The urine oligosaccharide profiles from healthy controls and patients diagnosed with different lysosomal storage diseases (fucosidosis, α-mannosidosis, GM1 gangliosidosis, GM2 gangliosidosis type Sandhoff, glycogen storage disease type 2 and 3, aspartylglucosaminuria, Schindler disease and galactosialydosis) and related disorders (glycogen storage diseases) are presented. In summary, these procedures are powerful tools for the rapid identification and characterization of these disorders. In all cases, the oligosaccharide profiles are strongly informative, identified the abnormal compounds, and are good alternatives to traditional screening tests that use thin layer chromatography. These tests have great sensibility and resolution and are automatable, enabling extended screening of these diseases, both as a first step in diagnosis or for confirming the pathogenicity of mutations that may be detected with next generation sequencing techniques.

Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13C6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12C6 'light' and 13C6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

Characterization of Cryptopygus antarcticus endo-β-1,4-glucanase from Bombyx mori expression systems.

Endo-β-1,4-glucanase (CaCel) from Antarctic springtail, Cryptopygus antarcticus, a cellulase with high activity at low temperature, shows potential industrial use. To obtain sufficient active cellulase for characterization, CaCel gene was expressed in Bombyx mori-baculovirus expression systems. Recombinant CaCel (rCaCel) has been expressed in Escherichia coli (Ec-CaCel) at temperatures below 10°C, but the expression yield was low. Here, rCaCel with a silkworm secretion signal (Bm-CaCel) was successfully expressed and secreted into pupal hemolymph and purified to near 90% purity by Ni-affinity chromatography. The yield and specific activity of rCaCel purified from B. mori were estimated at 31 mg/l and 43.2 U/mg, respectively, which is significantly higher than the CaCel yield obtained from E. coli (0.46 mg/l and 35.8 U/mg). The optimal pH and temperature for the rCaCels purified from E. coli and B. mori were 3.5 and 50°C. Both rCaCels were active at a broad range of pH values and temperatures, and retained more than 30% of their maximal activity at 0°C. Oligosaccharide structural analysis revealed that Bm-CaCel contains elaborated N- and O-linked glycans, whereas Ec-CaCel contains putative O-linked glycans. Thermostability of Bm-CaCel from B. mori at 60°C was higher than that from E. coli, probably due to glycosylation.

Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure-function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

A Highly Efficient and Visualized Method for Glycan Enrichment by Self-Assembling Pyrene Derivative Functionalized Free Graphene Oxide

Protein glycosylation plays key roles in many biological processes, such as cell growth, differentiation, and cell-cell recognition. Therefore, global structure profiling of glycans is very important for investigating the biological significance and roles of glycans in disease occurrence and development. Mass spectrometry (MS) is currently the most powerful technique for structure analysis of oligosaccharides, but the limited availability of glycan/glycoproteins from natural sources restricts the wide adoption of this technique in large-scale glycan profiling. Though various enrichment methods have been developed, most methods relay on the weak physical affinity between glycans and adsorbents that yields insufficient enrichment efficiency. Furthermore, the lack of monitoring the extent/completeness of enrichment may lead to incomplete enrichment unless repeated sample loading and prolonged incubation are adopted, which limits sample handling throughput. Here, we report a rapid, highly efficient, and visualized approach for glycan enrichment using 1-pyrenebutyryl chloride functionalized free graphene oxide (PCGO). In this approach, glycan capturing is achieved by reversible covalent bond formation between the hydroxyl groups of glycans and the acyl chloride groups on graphene oxide (GO) introduced by π-π stacking of 1-pyrenebutyryl chloride on the GO surface. The multiple hydroxyl groups of glycans lead to cross-linking and self-assembly of free PCGO sheets into visible aggregation within 30 s, therefore achieving simple visual monitoring of the enrichment process. Improved enrichment efficiency is achieved by the large specific surface area of free PCGO and heavy functionalization of highly active 1-pyrenebutyryl chloride. Application of this method in enrichment of standard oligosaccharides or N-glycans released from glycoproteins results in remarkably increased MS signal intensity (approximately 50 times), S/N, and number of glycoform identified.

O-Glycosylated IgA Rheumatoid Factor Induces IgA Deposits and Glomerulonephritis

Structural aberrations of O-linked glycans present in the IgA1 hinge region are associated with IgA nephropathy, but their contribution to its pathogenesis remains incompletely understood. In this study, mice implanted with hybridoma secreting 6-19 IgA anti-IgG2a rheumatoid factor, but not 46-42 IgA rheumatoid factor bearing the same IgA allotype, developed mesangial deposits consisting of IgA, IgG2a, and C3. Studies in immunoglobulin- and C3-deficient mice revealed that the development of these glomerular lesions required the formation of IgA-IgG2a immune complexes and subsequent activation of complement. The proportion of polymeric and monomeric forms, the IgG2a-binding affinity, and the serum levels of IgA-IgG2a immune complexes were similar between 6-19 IgA- and 46-42 IgA-injected mice. In contrast, the analysis of oligosaccharide structures revealed highly galactosylated O-linked glycans in the hinge region of 6-19 IgA and poorly O-glycosylated in the hinge region of 46-42 IgA. Furthermore, the structure of N-linked glycans in the CH1 domain was the complex type in 6-19 IgA and the hybrid type in 46-42 IgA. In summary, this study demonstrates the presence of O-linked glycans in the hinge region of mouse IgA and suggests that 6-19 IgA rheumatoid factor-induced GN could serve as an experimental model for IgA nephropathy.

Structural analysis of 1,3-linked galactose-containing oligosaccharides in Schizosaccharomyces pombe mutants harboring single and multiple -galactosyltransferase genes disruptions

In the fission yeast Schizosaccharomyces pombe, galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases in the lumen of the Golgi apparatus. In S. pombe, the major in vitro α1,2-galactosyltransferase activity has been purified, the gma12(+) gene has been cloned, and three α-galactosyltransferase genes (gmh1(+)-gmh3(+)) have also been partially characterized. In this study, we found three additional uncharacterized genes with homology to gmh1(+) (gmh4(+)-gmh6(+)) in the fission yeast genome sequence. All possible single disruption mutants and the septuple disruption strain were constructed and characterized. The electrophoretic mobility of acid phosphatase prepared from gma12Δ, gmh2Δ, gmh3Δ and gmh6Δ mutants was higher than that from wild type, indicating that Gma12p, Gmh2p, Gmh3p and Gmh6p are required for the galactosylation of N-linked oligosaccharides. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides from each single mutant showed that Gma12p, Gmh2p and Gmh6p are involved in galactosylation of O-linked oligosaccharides. The septuple mutant exhibited similar drug and temperature sensitivity as a gms1Δ mutant that is incapable of galactosylation. Oligosaccharide structural analysis based on HPLC and methylation analysis revealed that the septuple mutant still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating that an unknown α1,3-galactosyltransferase activity was still present in the septuple mutant.

Positive expressions of N-acetylglucosaminyltransferase-V (GnT-V) and β1-6 branching N-linked oligosaccharides in human testicular germ cells diminish during malignant transformation and progression

N-acetylglucosaminyltransferase-V (GnT-V) is an enzyme that catalyzes beta1-6 branching of N-acetylglucosamine on asparagines (N)-linked oligosaccharides of cell proteins. We examined the implication of GnT-V and beta1-6 branching N-linked oligosaccharide expression in human testicular germ cells during malignant transformation and cancer progression. We analyzed immuhistochemically orchiectomy specimens of 130 patients with testicular germ cell tumors (TGCT) using anti-GnT-V monoclonal antibody, and compared GnT-V expression with clinicopathological features. N-linked oligosaccharide structural analysis was also performed to confirm the oligosaccharide profile produced by GnT-V. GnT-V was positive in all normal testis samples. This positive incidence declined in TGCT according to clinical stage; 16/71 (22.5%) in stage I, and 3/59 (5.1%) in stage II/III (p=0.015, chi(2) test). When divided into pathological subtypes, GnT-V positive incidences in stage I seminoma, stage II/III seminoma, stage I non-seminomatous germ cell tumor (NSGCT), and stage II/III NSGCT were 3/43 (7%), 0/22 (0%), 13/28 (46.4%), and 3/37 (8.1%), respectively. In stage I NSGCT, patients with GnT-V-negative tumor samples were at a significantly higher risk of recurrence than those with GnT-V-positive tumors (p=0.015, log-rank test). N-linked oligosaccharide structural analysis revealed that a normal testis has three kinds of beta1-6 branching N-linked oligosaccharides, all of which are downregulated in TGCT tissues. These results suggest that GnT-V and beta1-6 branching N-linked oligosaccharide expressions are downregulated during carcinogenesis and progression of human TGCT. GnT-V may be a promising recurrence predictor for stage I NSGCT.

Fucosylation of N-Glycans Regulates the Secretion of Hepatic Glycoproteins into Bile Ducts

Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.

A Critical Role for Sialylation in Cryoglobulin Activity of Murine IgG3 Monoclonal Antibodies

Cryoprecipitating IgG3 autoantibodies have been shown to play a significant role in the development of murine lupus-like autoimmune syndrome. However, the structural basis of IgG3 cryoprecipitation still remains to be defined. In view of the implication of positively charged amino acid residues present in variable regions in IgG3 cryoglobulin activity, we explored the role of terminal sialic acids in oligosaccharide side chains for the cryogenic activity of IgG3 mAb. Comparative oligosaccharide structural analysis of different cryogenic and non-cryogenic IgG3 mAb showed an inverse correlation between the extent of sialylation and cryogenic activity. The inhibitory role of sialylation was further confirmed by the demonstration of enrichment of less and more sialylated IgG3 in cryoprecipitated and noncryoprecipitated fractions, respectively, separated from four different cryogenic IgG3 mAb. Significantly, the sialic acid contents of the latter fraction became comparable to those of non-cryogenic IgG3 mAb. Finally, we observed that highly sialylated non-cryogenic IgG3 mAb was more potent in the inhibition of cryoprecipitation of cryogenic IgG3 mAb. Our results thus suggest that the content of negatively charged sialic acids in oligosaccharide side chains is one of the critical factors to determine IgG3 cryoglobulin activity, along with amino acid sequences of the IgG3 variable regions.

Structural analysis of oligosaccharides by a 2 or 3-dimensional mapping method

Structural analysis of oligosaccharides by a 2 or 3-dimensional mapping method

Structural Analysis of Oligosaccharides from Lipopolysaccharide (LPS) of Escherichia coli K12 Strain W3100 Reveals a Link between Inner and Outer Core LPS Biosynthesis

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for approximately 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional beta-D-GlcN in 1-->7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L-alpha-D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an alpha-Kdo-(2-->4)-[alpha-l-Rha-(1-->5)]-alpha-Kdo-(2-->4)-alpha-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L-alpha-D-Hep-(1-->6)-alpha-D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.

Structural analysis of oligosaccharides by a combination of electrospray mass spectrometry and bromine isotope tagging of reducing-end sugars with 2-amino-5-bromopyridine.

Model reducing-end oligosaccharides were successfully labeled by a brominated aromatic amine reagent, 2-amino-5-bromopyridine (ABP), through reductive amination. Using either a combination of liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation or liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), sequence information corresponding to the model oligosaccharides was revealed with little ambiguity via the diagnostic unique twin peaks arising from the bromine isotopes, for both the molecular ions of the derivatized oligosaccharides and their fragments. No fragment ions arising from loss of the bromine atom were observed.

核磁気共鳴法を用いたオリゴ糖の構造解析

核磁気共鳴法を用いたオリゴ糖の構造解析

Analysis of Oligosaccharide Structures of Glycoproteins in Polyacrylamide Gel

Analysis of Oligosaccharide Structures of Glycoproteins in Polyacrylamide Gel

Quantitative analysis of oligosaccharide structure of glycoproteins

A sensitive and quantitative method for the structural analysis of oligosaccharide was established for the glycoform analysis of glycoproteins. In this study,N-linked oligosaccharides of human IgG and bovine transferrin were analyzed for the evaluation of the method. Carbohydrate moiety of glycoprotein was released by hydrazinolysis and purified by paper chromatography. The oligosaccharides were labeled with a fluorescent dye, 2-aminobenzamide, for the enhancement of detection sensitivity. Sialylated (acidic) oligosaccharides were separated from neutral oligosaccharide by employing a strong anion-exchange column (MonoO) followed by the treatment with sialidase. Enzymatically desialyated fractions and neutral fractions of oligosaccharides were applied to normal-phase HPLC to resolve the peaks according to glucose unit (GU). The structure of separated molecules was further determined by sequential digestion with exoglycosidases. As a result, disialylated biantennary complextype oligo saccharide was found to be a major sugar chain in bovine transferrin (63%). In human IgG, core fucosylated asialobiantennary complex oligosaccharides were dominant. These results coincided well with reported results.