Glycoprotein-derived Oligosaccharides Research Articles

Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans

An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the oligosaccharides shift to those of digested oligosaccharides. We examined β-galactosidase, α-mannosidase, β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, and found only β-galactosidase and α-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the reaction was completed by injecting a 3.6cm long plug of 200 and 50mU/mL concentration of exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10min when saccharide derivatives were achieved in the enzyme plug. The reaction of α-fucosidase and α-mannosidase was completed by decreasing the electrophoretic voltage to −2kV when the APTS oligosaccharides were passing through an exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of glycoprotein glycans.

Online Cleanup Liquid Chromatography for the Analysis of Glycoprotein-Derived Oligosaccharides Labeled with 7-Amino-4-methylcoumarin

Online Cleanup Liquid Chromatography for the Analysis of Glycoprotein-Derived Oligosaccharides Labeled with 7-Amino-4-methylcoumarin

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.

Application of Online Preconcentration Affinity Capillary Electrophoresis Method to Glycans Labeled with 8-Aminonaphthalene-1,3,6-trisulfonic Acid Using Blue Light Emitting Diode-induced Fluorescence Detection

An online preconcentration technique, large volume sample stacking with an electroosmotic flow pump, was combined with partial filling affinity capillary electrophoresis (PFACE) to create a highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with plant lectins. Oligosaccharides were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) for use in a blue light emitting diode-induced fluorescence detection capillary electrophoresis system. ANTS-labeled oligosaccharides were delivered to an entire neutrally coated capillary, and lectin solution was then hydrodynamically introduced from the outlet of the capillary as a short plug. When negative voltage was then applied, a low concentration sample solution caused a significant flow by electroosmosis from anode to cathode and the ANTS-labeled oligosaccharides moved quickly towards the anode and concentrated in the lectin phase. Finally, when the electroosmotic flow became negligible, ANTS-labeled saccharides passed through the lectin plug and were detected at the anodic end. The sensitivity was enhanced by a factor of roughly 200 compared to typical hydrodynamic injection (13.8 kPa, 5 s).

Partial filling affinity capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump for sensitive profiling of glycoprotein-derived oligosaccharides

An online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was combined with partial filling affinity capillary electrophoresis (PFACE) to realize highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with some plant lectins. Oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were delivered to an entire neutrally-coated capillary and then lectin solution was hydrodynamically introduced from the outlet of the capillary as a short plug. A negative voltage was then applied after immersion of both ends of the capillary in 100mM Tris–acetate buffer, pH 7.0 containing 0.5% hydroxypropylcellulose as electrophoresis buffers. A low concentration of electrolytes in the sample solution causes a significant flow by electroendosmosis from anode to cathode and the APTS-labeled oligosaccharides move quickly towards the anode and concentrate in the lectin phase. Finally, electroosmotic flow becomes negligible when the capillary is filled with the background electrolyte delivered from the anodic reservoir and APTS-labeled saccharides pass through the lectin plug and are detected at the anodic end. If the APTS-labeled oligosaccharides are recognized by the lectin, the migration profiles should be altered. The sensitivity was enhanced by a factor of ca. 900 compared to typical hydrodynamic injection (3.45kPa, 10s). By this method, increased residence time of APTS-saccharides in the lectin plug indicates highly efficient interaction with lectins, which differs completely from the results obtained by ordinary lectin PFACE. The run-to-run repeatability (n=18) of the migration time and peak area was high, with relative standard deviations of less than 0.7% and 6.1%, respectively.

Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis

Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N 4-( N-acetyl-β- d-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α 1-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3 h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100 fmol and 4 fmol, respectively.

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins ( Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.

Affinity Entrapment of Oligosaccharides and Glycopeptides Using Free Lectin Solution

Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol precipitation after dissolution in water. This method enabled group separation of 2-aminopyridine-labeled oligosaccharides derived from ovalbumin to galacto-oligosaccharides and agalacto-oligosaccharides by Ricinus communis agglutinin, and to high mannose- and hybrid-type oligosaccharides by wheat-germ agglutinin. Fractional precipitation based on differences in affinity for concanavalin A was accomplished by adding an appropriate concentration of methyl α-mannoside as an inhibitor. In the second method, tryptic digests of glycoproteins were mixed with a lectin solution, and the glycopeptide-lectin conjugates were specifically trapped on a centrifugal ultrafiltration membrane with cut-off of 10 kD. Trapped glycopeptides, as retentates, were passed through membranes by resuspension in diluted acid. This method is particularly useful for the enrichment of glycopeptides in protease digestion mixtures for glycosylation analyses by liquid chromatography-mass spectrometry.

Ion-pair chromatographic separation of glycoprotein derived oligosaccharides as their 8-aminopyrene-1,3,6-trisulfonic acid derivatives

A reversed-phase ion-pair gradient liquid chromatographic method has been developed for separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeled oligosaccharides derived from some glycoproteins. This separation mode has high capability for structural recognition of oligosaccharide isomers, which are usually difficult to separate using commonly used partition chromatography. In addition, some glycoprotein-derived complex-type oligosaccharides that have linkage isomers in one lactosamine arm and neuraminic acids existing in fetuin, as well as differences in the numbers of lactosamine branches, are separable using this mode. The ion-pair mode is promising for simple analyses of glycoprotein-derived oligosaccharides that are labeled with APTS.

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60–360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of α1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower p I values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher p I values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

Binding specificities of lectins to immobilized glycoproteins and oligosaccharides differ from those of immobilized lectins to oligosaccharides.

The carbohydrate-binding specificities of lectins in solution to glycoproteins and neoglycolipids immobilized on a solid phase were analyzed in order to establish a simple, rapid method for structural analysis of the carbohydrate moieties of small amounts of individual glycoproteins blotted on membrane. Eight glycoproteins containing typical O-linked tetrasaccharides or a series of typical N-linked oligosaccharides of the high-man-nose type, hybrid type, and complex type and 6 neoglycoproteins containing mono- or di-saccharides were dot blotted on membranes and the membranes were then reacted with 8 kinds of horseradish peroxidase-conjugated lectins before and after heat treatment. Neoglycolipids containing the glycoprotein-derived oligosaccharides immobilized on a thin layer chromatography plate were also reacted with lectins. The heat treatment of the membrane increased lectin reactivity toward the glycoproteins. The carbohydrate-binding behavior of lectins, Phaseolus vulgaris erythroagglutinin, wheat germ agglutinin, and concanavalin A in solution toward glycoproteins and neoglycolipids immobilized on a solid phase differed from that of immobilized lectins toward oligosaccharides in solution. This difference should be noted in lectin detection of specific carbohydrates of individual glycoproteins on membrane.

Sulfate Detection in Glycoprotein-Derived Oligosaccharides by Artificial Neural Network Analysis Fourier-Transform Infrared Spectra

We report the use of an artificial neural network to analyze the fingerprint region of Fourier-transform infrared (ir) spectra of oligosaccharides for the presence of sulfate groups. This assay can rapidly and nondestructively detect the presence of sulfate in as little as 1 nmol (≍2 μg) of a glycoprotein-derived monosulfated decasaccharide. The neural network was trained to recognize the presence of sulfate groups by presenting it with 45 ir spectra of sulfated and nonsulfated mono- and oligosaccharides. No prior knowledge of the characteristic ir spectral features of a sulfate group was needed as input. The training process required between 3 and 10 h, while analysis of a spectrum with the trained neural network requires only 0.1 s.

A novel method for the incorporation of glycoprotein-derived oligosaccharides into neoglycopeptides.

We describe a new method for the transfer of carbohydrate moieties to polypeptides in which complex carbohydrate, in the form of glycosyl amino acid, is removed from an available glycoprotein, derivatized, and reacted with a polypeptide via an iodoacetylated alpha-amino group. A family of oligomannose chains, N-linked to the side chain of Asn, was obtained from ovalbumin by pronase digestion and purified as previously described. A reactive sulfhydryl group was specifically placed on these molecules by reaction of 2-iminothiolane with the Asn alpha-amino group. Separately, the alpha-amino group of the peptide GGYR was specifically iodoacetylated by reaction with iodoacetic anhydride at pH 6. Reaction of the thiol-containing carbohydrate with iodoacetylated peptide at pH 8 gave in high yield the corresponding oligomannosyl-peptides, whose structures were confirmed by mass spectrometry. A peptide inhibitor of HIV protease was also oligomannosylated by this procedure. The principle advantage of this method is the efficiency of the reaction even when performed with stoichiometric amounts of the two molecules at low concentration. It should be feasible to extend this chemistry to larger polypeptides.

Calculated two-dimensional sugar map of pyridylaminated oligosacchardies: Elucidation of the jack bean α-mannosidase digestion pathway of Man 9GlcNAc 2

We have developed a two-dimensional (2-D) mapping of puridylaminated oligosaccharides as an aid to structural determination of glycoprotein-derived oligosaccharides. Using the available data of reverse-phase HPLC of pyridylamino-oligosaccharides, this was further extended to parameterization of unit contribution by each sugar component, which allows the prediction of possible structures from the elution volume. We have extended this approach to the data obtained with amide-silica HPLC column to obtain a calculated 2-D mapping technique for the oligomannose-type oligosacharides (M-series). In this method, the elution volumes of all possible pyridylamino-oligosaccharides up to the size of Glc 1Man 9GlcNAc 2 (50 in total) are calculated from the established UC values to construct a 2-D map. To test the validity of the calculated 2-D map, the structures of intermediate PA-oligosaccharides generated during the α-mannosidase (jack bean) digestion of Man 9GlcNAc 2 (porcine thyroglobulin) were analyzed to establish the digestion pathway. The validity of this approach is substantiated by an independent deduction of the intermediate structures based on structural relationships and the coincidence of elution volumes. Our results agree well with the recently published digestion pathway of Man 5GlcNAc 2 by the same enzyme and that of Man 9GlcNAc 2 by lysosomal α-mannosidase.

Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest

Carbohydrate characterization of recombinant glycoproteins entails determination of the primary structures and points of attachment of the oligosaccharide moieties. This article reviews several methods for oligosaccharide- and glycosylation-site characterization. A major recent advance in carbohydrate analysis has been the use of high-pH anion exchange (HPAE) chromatography for separation of glycoprotein-derived oligosaccharides. These separations are sensitive to molecular size, carbohydrate composition, linkage positions, and anomeric configurations. As a result, HPAE chromatography is a powerful technique for glycoprotein characterization and can serve as the basis of an "oligosaccharide map." Characterization of potential N-glycosylation sites involves determining whether each potential site is glycosylated, the extent of oligosaccharide processing at each site, and ideally, a detailed description of the distribution of oligosaccharides at each site. Several approaches to characterizing glycosylation sites are described, including peptide mapping and mass spectrometry. Treatment of a glycoprotein with endo-beta-N-acetylglucosaminidase H (endo H) followed by peptide:N-glycosidase F (PNGase F) can be used to distinguish sites that are not glycosylated from those carrying high-mannose structures and from those containing attached complex oligosaccharides. After individual glycosylation sites have been labeled by this series of reaction, the resulting peptides are characterized by automated Edman degradation. This technique is particularly valuable for characterizing peptides that contain more than one potential N-glycosylation site. An example is also given in which HPAE chromatography is used in conjunction with reversed-phase high-performance liquid chromatography tryptic mapping to obtain detailed information on the distribution of oligosaccharides at individual glycosylation sites.

Analysis of glycoprotein-derived oligosaccharides by high-pH anion-exchange chromatography

The technique of high-pH anion-exchange chromatography with pulsed amperometric detection has recently been shown to be a powerful method for resolving closely related oligosaccharides [M. R. Hardy and R. R. Townsend, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3289–3293]. This report describes separations involving a total of nineteen different high-mannose, hybrid and complex-type oligosaccharides isolated after peptide:N-glycosidase F (PNGase F) or endo-β-N-acetylglucosaminidase H digestion of glycoproteins. Separations were carried out at a constant base concentration (0.1 M NaOH) using linear gradients from 0 to 0.2 M sodium acetate. The applicability of this chromatography for profiling the N-linked oligosaccharides of glycoproteins was demonstrated by generating “oligosaccharide maps” of PNGase F-liberated oligosaccharides from recombinant human tissue plasminogen activator, ribonuclease b, human transferrin, and bovine fetuin. Methods for recovering salt-free oligosaccharides after this chromatography were also investigated. On-line ion suppression with an anionic micromembrane suppressor cartridge was found to be capable of effective desalting up to a total sodium ion concentration of 0.15–0.2 M at a flow-rate of 1 ml/min. After high-pH anion-exchange chromatography with ion suppression, collected oligosaccharides were analyzed by fast-atom bombardment mass spectrometry after conversation to permethyl derivatives or after reductive amination with ρ-aminobenzoic acid ethyl ester.

Separation of neutral oligosaccharides by high-performance liquid chromatography

We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H 2O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of 3H by reduction with NaB 3H 4 permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides.

A rapid method for chromatographic analysis of oligosaccharides on Bio-Gel P-4

We have developed a method for the rapid analysis of oligosaccharides and their degradation products by chromatographic separation on Bio-Gel P-4 (−400 mesh). Flow rates of 64 ml cm −2/h −1 can be attained at 55–100 psi with either a single column of 0.6 × 100 cm or two coupled columns each 0.6 × 100 cm, permitting analyses to be carried out in 1.5 or 3.0 h, respectively. Differences of 1 glucose equivalent can be precisely distinguished for oligomers at least as large as 15 glucose equivalents. The applicability of this method for the analysis of glycoprotein-derived oligosaccharides is demonstrated. By assigning a value of 1 glucose equivalent per neutral sugar residue and 2 glucose equivalents per amino sugar residue in such oligosaccharides, the effect of various exo- and endoglycosidase digestions can be rapidly and precisely monitored. These columns are extremely stable, providing continuous operation for at least 9 months with over 300 sample applications without deterioration.

Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography

A thin-layer chromatography (tlc) system has been developed for the separation of glycoprotein-derivedoligosaccharides. The method involves chromatography on silica gel using n -propanol/acetic acid/water (3:3:2 v/v) as the solvent. This tlc method was used to separate pathological oligosaccharides isolated from individuals with G M1 gangliosidosis and with neuraminidase deficiency. The results indicate the potential usefulness of the system in the analysis of complex carbohydrates.