Effect of the quantity and linkage position of mannose (alpha 1,2) residues in capillary gel electrophoresis of high-mannose-type oligosaccharides.
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Carbohydrate Conformation↗
Biomedical subjects
A Guttman
Publications and source records attributed to A Guttman.
High-resolution capillary gel electrophoresis of reducing oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonate.
High-resolution capillary gel electrophoresis was used for the separation of oligosaccharides labeled with a novel fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) at the reducing termini by reductive amination. The APTS-saccharide adducts were detected by laser-induced fluorescence with excitation by the 488-nm Ar-ion laser and a 520-nm emission filter. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to each molecule of oligosaccharide. Derivatization parameters, such as labeling reagent concentration, labeling temperature, and time as well as the influence of reaction solvent are thoroughly discussed. Desialylation of several sialylated oligosaccharides with different structures and linkages due to the effects of labeling temperature and time are also addressed. Employing the optimized conditions suggested in this paper, fluorophore labeling efficiency greater than 97% was achieved with no significant loss of sialic acid residues. The fluorescently labeled oligosaccharides are then separated and quantified by capillary gel electrophoresis. Practical examples of low-level derivatization and high-resolution capillary gel electrophoresis separation of N-linked glycans of ribonuclease-B and fetuin are also shown.
Acetylglucosamine↗
Acid-catalyzed reductive amination of aldoses with 8-aminopyrene-1,3,6-trisulfonate.
The reductive amination of monosaccharides with 8-aminopyrene-1,3,6-trisulfonate (APTS) in seven different organic acids including the commonly used acetic acid was investigated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. The correlation between the yields of the saccharide-APTS adducts and pKa of the organic acid catalyst is consistent with general acid catalysis of the rate-determining step of the reductive amination reaction. Derivatization in the presence of organic acids of higher strength than acetic acid produced substantially higher yields of APTS-sugar adducts, an effect which is more pronounced for N-acetylamino sugars. Optimum yields were obtained using citric acid as a catalyst. Conversion of a few nanomoles of neutral saccharides to the APTS derivatives is achieved at 75 degrees C in less than 60 min.
Acids↗
Separation of 1-aminopyrene-3,6,8-trisulfonate-labeled asparagine-linked fetuin glycans by capillary gel electrophoresis.
Asparagine-linked glycans of bovine fetuin were separated by capillary gel electrophoresis after enzymatic release (peptide-N-glycosidase F) and labeling via reductive amination by a fluorescent dye, 1-aminopyrene-3,6-8-trisulfonate (APTS). At low separation pH (2.5) only two dominant peaks were observed. Increasing the separation buffer pH to 4.75 resulted in complete separation of two primary doublets and several minor peaks from the fetuin N-linked glycan pool. Two of the four major peaks were spiked with purified individual standards and were identified as trisialylated triantennary structures with different sialylation linkages. The other two larger peaks were postulated to be tetrasialylated triantennary structures, based on calculations considering their corresponding glucose unit (GU) values. Effects of the electrophoretic separation parameters, such as gel concentration, electric field strength and temperature on the migration behavior of the two major doublets of the fetuin glycan pool were also thoroughly examined. Our data suggest that the capillary gel electrophoresis separation of the multisialylated branched oligosaccharides with different linkage isomers, released from bovine fetuin, is fundamentally based on their degree of sialylation and hydrodynamic volumes.
Asparagine↗
Capillary sodium dodecyl sulfate-gel electrophoresis of proteins.
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Electrophoresis, Capillary↗
Capillary gel electrophoresis.
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Amino Acids↗
Rapid chiral separation methods development by cyclodextrin-mediated capillary electrophoresis for acidic and basic compounds.
Chiral separation methods development using conventional techniques such as GC or HPLC requires a lot of experience, effort, and expense, due to the wide diversity of the optically active solutes and their possible chiral selectors. Capillary electrophoresis has received increased attention as an alternative technique for chiral separation due to its inherent high efficiencies and ease of methods development. However, due to the wide variety of chiral selectors available in CE, the benefits of this technique might be diminished without an appropriate methods development scheme. In this paper detailed examples are shown for fast, efficient, and predictable chiral capillary electrophoresis separation methods development based on a new and systematic theory. Optimized separations and their parameters are presented for several enantiomeric acids and bases. All the three possible cases, such as the use of low and high pH, as well as pH = pK buffer systems are thoroughly discussed.
Acids↗
Capillary and slab gel electrophoresis profiling of oligosaccharides.
Capillary electrophoresis with laser-induced fluorescent detection, a one-dimensional version of the well-established planar analytical method of polyacrylamide gel electrophoresis, has been proven to be a powerful new microanalytical method for profiling complex carbohydrates. In this paper a comparison is presented between the planar high concentration polyacrylamide gel electrophoresis method and capillary electrophoresis of different carbohydrates with respect to performance and efficiency. N-Linked oligosaccharides were released from several glycoproteins, including fetuin, human immunodeficiency virus (HIV) envelope recombinant glycoprotein (GP-120), alpha 1-acid glycoprotein and ribonuclease B, using recombinant peptide-N-glycosidase F (PNGase F). Both separation methods involve labeling of the released carbohydrates at the reducing end with the fluorescent dye, disodium 8-amino-1,3,6-naphthalene trisulfonate (ANTS). Fluorophore labeling was followed by separation of the labeled oligosaccharides either by high concentration polyacrylamide gel electrophoresis or capillary electrophoresis.
Animals↗
Novel separation scheme for capillary electrophoresis of enantiomers.
A systematic approach is described for methods development of chiral separations of weak acidic and basic compounds by capillary electrophoresis, using several natural and derivatized neutral cyclodextrins as chiral selectors. Following the methods development scheme suggested here, the appropriate pH of the running buffer as well as the type and concentration of the cyclodextrin is established for the separation of enantiomers. Preselected chiral selectors of beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin and dimethyl-beta-cyclodextrin in low and high concentrations, dissolved in low pH, high pH or pH = pK buffers, are employed during the separation method development and optimization. Depending on the type of separation, introduced by Vigh (desionoselective: only the nondissociated; ionoselective: only the dissociated; duo-selective: both enantiomers complex selectively), in most instances at least one of the pH/cyclodextrin combinations results in acceptable separation of the solute enantiomers. The viability of the approach is demonstrated through step by step development of chiral separation for several basic and acidic enantiomers.
Aspartic Acid↗
Capillary gel electrophoresis separation of high-mannose type oligosaccharides derivatized by 1-aminopyrene-3,6,8-trisulfonic acid.
In this paper we report the capillary gel electrophoresis separation of 1-aminopyrene-3,6,8-trisulfonic acid (APTS) labeled oligosaccharides, released enzymatically from bovine pancreatic ribonuclease B. The released and labeled high-mannose structures were identified by spiking the separated peaks with the appropriate commercially available individual oligosaccharides. Baseline separation of the three positional isomers of the mannose-7 and mannose-8 oligosaccharides was attained. Comparison of the electrophoretic mobilities of the high-mannose type branched carbohydrates to the linear molecules of maltooligosaccharides (glucose oligomers) have been shown using different gel concentrations in the running buffer system. We observed that increasing gel concentration in the running buffer causes an increase in the relative mobility values of the high-mannose type carbohydrate molecules compared to the linear glucose oligomers. Analysis of our data indicated that this increase in relative migration time was not due to sieving, but seemed to be related to the mannose content and hydrodynamic volume of the branched glycans as well as to the viscosity of the separation medium.
Animals↗
On the separation mechanism of capillary sodium dodecyl sulfate-gel electrophoresis of proteins.
Polyethylene oxide-mediated capillary sodium dodecyl sulfate-electrophoresis is a recently established, high-resolution separation method for fast purity check and molecular mass assessment of protein molecules. The effects of the sieving polymer chain length and concentration on the separation mechanism of sodium dodecyl sulfate-protein complexes were examined. The studies aimed to clarify whether the separation can be described by either the Ogston sieving theory, or the reptation or reptation-with-stretching theory. Polyethylene oxides with molecular masses of 100,000, 300,000 and 900,000 Da were used as separation matrices at various concentrations ranging from 1-4%, 0.5-2% and 0.25-1%, respectively. The separation phenomena was examined using a standard protein test mixture containing six proteins in the molecular mass range of 14,200-97,400 Da. A possible separation mechanism of reptation with stretching is suggested, where separation performance was improved with increasing sieving polymer chain lengths and/or concentration.
Electrophoresis, Polyacrylamide Gel↗
Capillary electrophoresis separation of oligosaccharides: I. Effect of operational variables.
The influence of operating variables on the separation of labeled oligosaccharide molecules in capillary electrophoresis with polymer networks is presented. In this study, an equation was derived that relates the effects of electrophoresis variables such as field strength, temperature, molecular weight and gel concentration to the migration velocity. As a model system, 8-aminonaphthalene-1,3,6-trisulfonate ANTS-labeled wheat starch digest was examined to show the validity of the equation. As an illustration of the high resolving power achievable with capillary electrophoresis, oligosaccharide molecules up to the degree of polymerization of 25 were separated with the very high efficiency of N > 10(6) and efficiency generation rate of 3500 plates/m/s for maltose. Separation based on the size of the oligosaccharides was not obtained with either entangled or unentangled polymer solutions.
Electric Conductivity↗
Effect of operational variables on the separation of proteins by capillary sodium dodecyl sulfate-gel electrophoresis.
Capillary sodium dodecyl sulfate (SDS)-gel electrophoresis, for fast and high-resolution separations of protein molecules based on their molecular masses was investigated, employing a polymeric sieving network of polyethylene oxide (PEO). A standard mixture of five proteins ranging from 14.2-66.0 kDa in molecular mass was used to evaluate the sieving matrix. A general migration velocity equation was derived for capillary SDS-gel electrophoresis of proteins and supported by the experimental data. This equation gives a better understanding of SDS-protein separations via capillary gel electrophoresis. Results are presented regarding the effects of different operational variables such as gel concentration, electric field strength, molecular mass, and temperature on the electrophoretic migration properties of the different size protein molecules.
Electromagnetic Fields↗
Comparison of the separation of proteins by sodium dodecyl sulfate-slab gel electrophoresis and capillary sodium dodecyl sulfate-gel electrophoresis.
Capillary sodium dodecyl sulfate-gel electrophoresis, a one-dimensional version of the well-established planar analytical method of SDS-polyacrylamide gel electrophoresis, has proven a powerful new microanalytical method for the separation of protein molecules according to their size. In this paper the planar SDS-PAGE and the capillary SDS-gel electrophoresis of different proteins are compared with respect to performance, precision, and efficiency. Molecular weight versus migration distance and migration time curves for 65 proteins, running from 14,200 to 205,000 Da, are given for both methods.
Capillary Action↗
Protein gel analysis by capillary electrophoresis.
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Capillary Action↗
High-performance capillary electrophoresis of SDS-protein complexes using UV-transparent polymer networks.
This paper demonstrates the use of UV-transparent replaceable polymer networks for the separation of SDS-protein complexes on the basis of molecular weight. First, the use of linear (i.e. non-cross-linked) polyacrylamide is shown to provide molecular separation of SDS-protein complexes. A study reveals such columns to yield significantly greater lifetime than cross-linked gels because of the flexibility of the noncovalently attached polymer chains. However, column lifetime was still found to be limited (approximately 20-40 injections), and detection at 214 nm was problematical because of the absorbance of polyacrylamide. UV-transparent polymer networks of dextran and PEG were substituted for polyacrylamide with successful molecular weight sieving of SDS-protein complexes at 214 nm. Due to their low to moderate viscosities, these networks could be routinely replaced leading to the possibility of hundreds of injections with a single column. Migration time reproducibilities of 0.5% RSD or less were found with replacement of the network. Using dextran, calibration plots of peak area vs concentration of standard protein were linear over the range of 0.5 microgram/mL up to at least 0.25 mg/mL. Furthermore, plasma samples could be directly utilized because of the strong solvating power of SDS. Rapid separation of protein mixtures are demonstrated with these UV-transparent polymer networks.
Dextrans↗
Influence of pH on the migration properties of oligonucleotides in capillary gel electrophoresis.
The effect of pH on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary gel electrophoresis was investigated. Different homooligodeoxyribonucleotides of equal chain length showed significant differences in relative migration when the pH of the gel buffer was varied from pH 6 to 8, parallel with the running buffer. A similar variation in migration order was observed during the electrophoretic equilibration of a pH 8 gel-filled capillary column with a pH 6 running buffer. In the latter instance, the current reached the new level after 20 min of electrophoretic equilibration with the pH 6 running buffer. However, it was observed that the migration order characteristic of the pH 6 gel was achieved only after 4 h of electrophoretic equilibration. To avoid this time-consuming equilibration process, these results suggest that gel-filled capillary columns should be prepared with the same buffer (composition and pH) that will be used as the running buffer during the separations.
Buffers↗
Prediction of migration behavior of oligonucleotides in capillary gel electrophoresis.
The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary polyacrylamide gel electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lengths but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide capillary gel electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10-20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.
Base Sequence↗