Analysis of carbohydrate-mediated heterogeneity and characterization of N-linked oligosaccharides of glycoproteins by high performance capillary electrophoresis.

Capillary zone electrophoresis (CZE) has been investigated as an alternative method to analyze the carbohydrate moieties of glycoproteins. Carbohydrate-mediated microheterogeneity of the recombinant plasminogen activator (rt-PA) was examined. The glycoprotein was resolved in multiple electrophoretic species using CZE but the separation was complicated by adsorption of the molecules to the wall of the capillary. The influence of several parameters, such as pH, molarity of the buffer and addition of a cationic additive, on the separation of glycopeptides was investigated. High resolution and reproducible separations of rt-PA glycopeptides carrying hybrid and complex type chains were obtained using either a 100 mM phosphate buffer, pH 6.6, or a 100 mM Tricine buffer, pH 8.2, containing 1.25 mM of putrescine. N-Oligosaccharides from fetuin, t-PA and alpha 1-acid glycoprotein were separated within 20 min on the basis of both their sialic acid content and their structure. The use of an oligosaccharide fingerprinting technique, such as the present one, could have many applications in biotechnology to assess, for example, the consistency of production of a glycoprotein or for analytical glycoprotein chemistry.     

Chiral separation by capillary electrophoresis with oligosaccharides.

Maltodextrins, i.e., mixtures of linear alpha-(1-4)-linked D-glucose polymers, were found to be effective as chiral electrolyte modifiers to perform direct, rapid separations by capillary electrophoresis of racemic mixtures of 2-arylpropionic acid non-steroidal anti-inflammatory compounds and coumarinic anticoagulant drugs, and also diastereomeric cephalosporin antibiotics. Enantioselectivity seemed to be dependent on an as yet unidentified combination of variables.     

Resolution of structural isomers of sialylated oligosaccharides by capillary electrophoresis

The resolution of structural isomers in mixtures of oligosaccharides is often challenging. Capillary electrophoresis was employed to separate three sets of structural isomers of sialylated oligosaccharides found in human milk and bovine colostrum. Different running buffers were necessary to achieve optimal baseline resolution. To resolve 3'- and 6'-sialyllactoses, 0.2 M aqueous sodium phosphate containing 40% methanol as an organic modifier was used as a running buffer. To resolve 3'- and 6'-sialyllactosamines, 0.4 M aqueous sodium phosphate without organic modifier was used. Baseline resolution of sialyllacto-N-tetraose-a and -b and sialyllacto-N-neotetraose-c was achieved with a 0.4 M Tris-HCl buffer containing 250 mM sodium dodecyl sulfate and 10% methanol as the organic modifier. Thus, each of these sets of structural isomers of sialylated oligosaccharides required a unique running buffer with respect to buffer type, concentration, pH, presence of organic modifiers, and surfactants. Similar electrophoresis conditions may be useful for resolving and analyzing other structural isomers of acidic oligosaccharides by capillary electrophoresis.     

Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence

A method, using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for analyzing chitin oligosaccharides is described. Chitin oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) via reductive amination at 37 degrees C for 16 h (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid-phosphate buffer or an alkaline borate buffer. The effects of buffer types, buffer pH values, and buffer concentrations on the separation were examined. The analytes were successfully separated by using a pH 4.6 citric acid-phosphate within 19 min. The APTS-derivatized chitin monosaccharide (D-glucosamine) migrated first. The analytes were also completely separated by using a pH 9.0 borate buffer within 24 min. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized APTS labeling procedure and the CE-LIF method was demonstrated.     

Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis

Carbohydrate chains in glycoprotein pharmaceuticals have important roles for the expression of their biological activities. Therefore, development of an assessment method for the carbohydrate chains is an important parameter for quality control of glycoprotein pharmaceuticals such as newly developed therapeutic antibodies. In this report, we applied capillary electrophoresis with laser-induced fluorescence detection to the analysis of carbohydrate chains after releasing with glycoamidase followed by derivatization with 3-aminobenzoic acid. We found that four major oligosaccharides present in antibody pharmaceuticals were successfully separated with good resolution. The present method showed good precision in both migration times and relative peak areas, and gave comparable accuracy with that using a derivatization method with 8-aminopyrene-1,3,6-trisulfonate.     

Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis

We report on microbore liquid chromatography (microLC) and capillary electrophoresis (CE) separation of glycopeptides and high-mannose-type oligosaccharides, digested from recombinant phospholipase C, expressed in Pichia pastoris. The glycopeptides were subject to microLC/electrospray ionization/mass spectrometry (ESI-MS) and microLC/ESI-tandem MS (MS/MS) analysis that revealed high-mannose structure size variation between Man(7)GlcNAc(2) and Man(14)GlcNAc(2). Then, high-performance CE was applied to identify possible positional isomers of the high-mannose structures. For the CE experiments, the oligosaccharides were released from the glycoproteins by peptide-N-glycosidase F and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS). Excellent separation of the possible positional isomers was attained, suggesting one for Man(9)GlcNAc(2), two for Man(10)GlcNAc(2), three for Man(11)GlcNAc(2), Man(12)GlcNAc(2), and Man(13)GlcNAc(2), and two for Man(14)GlcNAc(2). The CE results provided complementary information to the microLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.     

Highly sensitive capillary electrophoresis analysis of N-linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser-induced fluorescence detection

We describe a highly sensitive CE with laser-induced fluorescence (LIF) detection for the analysis of N-linked oligosaccharides in glycoproteins using rhodamine 110 as a fluorescence derivatization reagent. One CE separation is performed using a fused-silica capillary and neutral pH buffer conditions and allows for the separation of sialo-oligosaccharides according to the number of sialic acids. An alternate separation is performed using the same capillary and acidic pH buffer conditions, enabling the separation of asialo-oligosaccharides according to their sizes. The derivatization and separation conditions for the analysis of sialo- and asialo-oligosaccharides were optimized. Furthermore, we applied the proposed method for the analyses of N-linked sialo- and asialo-oligosaccharides in glycoproteins (ribonuclease B, fetuin, and recombinant human erythropoietin).     

Analysis of glycoproteins, glycopeptides and glycoprotein-derived oligosaccharides by high-performance capillary electrophoresis

Recent developments in the analysis of glycoproteins by high-performance capillary electrophoresis are reviewed, with emphasis on their carbohydrate chains. Glycoforms of glycoproteins were directly separated from each other by careful optimization of the analytical conditions. Glycopeptides in tryptic digests were separated and the peptides carrying glycosylation sites were differentiated from others. Released oligosaccharide chains were separated at a low wavelength. Precolumn derivatization by various methods extended the utility of both the absorption at a low wavelength. Precolumn derivatization by various methods extended the utility of both the separation mode and detection technique. Dual mode analysis after derivatization permitted reliable identification and quantification without references.     

Protein mapping of peanut extract with capillary electrophoresis

Protein separation can be achieved with different modes of capillary electrophoresis, such as with capillary gel electroporesis (CGE) or with capillary zone electrophoresis (CZE). CZE protein mapping of peanut extract was approached in four different ways, combining neutral-coated or multilayer-coated capillaries with pHs well over or under the isoelectric point range of the proteins of interest. At acidic pHs, the mobility ranges of the major peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 were identified. Although the pH is a major factor in CZE separation, buffers with different compositions but with the same pH and ionic strength showed significantly different resolutions. Different components of the electrolyte were studied in a multifactorial design of experiment. CE-SDS and CZE proved to be suitable for protein mapping and we were able to distinguish different batches of peanut extract and burned peanut extract.     

On-line system for peptide mapping by capillary electrophoresis at sub-micromolar concentrations

Peptide mapping has been widely used for the identification of modified proteins involved in certain diseases. Despite the fact that capillary electrophoresis (CE) has been shown to be a powerful tool for the separation and detection of tryptic peptide fragments after protein digestion, this technique lacks sensitivity for mapping proteins isolated in very small quantities from biological samples. Consequently, it has been necessary to preconcentrate the protein before adding the proteolytic enzyme for digestion in solution. These experimental steps are quite long, labor intensive and require a lot of sample handling. In this paper, we describe an on-line system allowing digestion of the protein, followed by preconcentration, separation and detection of the tryptic fragments in 4 h. Up to an 800-fold preconcentration factor was achieved for cytochrome c, despite a loss of separation efficiency induced by the multiple-valve design of the system and dispersion of the 60-nl desorption plug. Moreover, our system showed good migration time reproducibility between peptide maps and could be reused for several samples.     

Preparative purification of tyrosinamide N-linked oligosaccharides

N-linked oligosaccharides from glycoproteins can be either analyzed on a sub-nanomole scale or preparatively purified on a multi-micromole scale. Each goal necessitates a unique analytical strategy often involving oligosaccharide derivatization to enhance separation and detection. Tyrosinamide-oligosaccharides were developed to facilitate the preparative purification of N-linked oligosaccharides. These have found many uses in oligosaccharide remodeling, in the preparation of neoglycoconjugates, in developing receptor probes, and even as analytical standards in chromatography. This review discusses progress in the preparation of tyrosinamide-oligosaccharides from different glycoproteins and their utility in glycobiology research.     

Analysis of underivatized cellodextrin oligosaccharides by capillary electrophoresis with direct photochemically induced UV‐detection

This work focuses on the development of a CE method allowing, for the first time, the simultaneous separation of the underivatized first seven cellodextrin oligomers (glucose, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose), with a view to analyze the hydrolysates obtained after partial acid depolymerization of nitrocellulose, and eight carbohydrates (ribose, xylose, fructose, mannose, galactose, maltose, lactose, and sucrose), which might be potential interfering compounds in explosives samples. Separation was achieved with a highly alkaline BGE containing sodium chloride and direct mid‐UV‐absorbance detection was performed after photo‐oxidation in the detection window. EOF was reversed to speed up the analysis using a dynamic capillary coating by hexadimethrine bromide. A central composite design was carried out to determine the effects of BGE conductivity and sodium hydroxide concentration on resolutions between neighboring peaks, and analysis time. A desirability analysis on modeled responses was applied to maximize resolutions and to minimize analysis time. The simultaneous analysis in 20 min total runtime of the 15 carbohydrates plus internal reference (naphthalene sulfonate) was carried out at 25°C with a BGE composed of 77.4 mM NaOH and 183 mM NaCl to adjust the conductivity at the optimum value. Finally, the resolution robustness was checked. This new method should also be of interest to monitor food and nonfood crop products.     

Haplotyping by capillary electrophoresis

The investigation of the genetic background and phenotype structures of complex diseases, such as cardiovascular or psychiatric disorders and tumors, is one of the most scrutinized fields of the post genomic era. Besides the multiplex analysis of genetic markers and polymorphisms throughout the whole genome, more and more attention is focused on the interaction between the etiological factors of these traits. Haplotype determination, rather than multiplex genotyping seems to be one of the first building blocks of this endeavor. This review focuses on the importance and theoretical background of haplotyping, and summarizes the recent examples of novel and emerging haplotyping techniques by capillary gel electrophoresis based DNA fragment analysis, a powerful tool for the examination of the inheritance of complex traits.     

Identification of hyaluronic acid oligosaccharides by direct coupling of capillary electrophoresis with electrospray ion trap mass spectrometry

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments.     

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 alpha1-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 pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI 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.     

Three-dimensional mapping of N-linked oligosaccharides using anion-exchange, hydrophobic and hydrophilic interaction modes of high-performance liquid chromatography

To determine the structure of N-linked oligosaccharides, a three-dimensional (3-D) sugar mapping technique for pyridylaminated neutral and sialyl oligosaccharides is proposed. The pyridylaminated oligosaccharide mixture is first separated by HPLC on a diethylaminoethyl anion-exchange column and the elution data are placed on the Z-axis. Neutral and mono-, di-, tri- and tetrasialyloligosaccharides are then individually separated on both a hydrophobic octadecylsilylsilica column and a hydrophilic amide-silica column under the same conditions as described previously for neutral oligosaccharides. The validity of the 3-D mapping technique was tested using sialyl pyridylaminated oligosaccharides from human serum glycoproteins.     

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.