Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ?M - H(-) ions.     

Activation energy associated with the electromigration of oligosaccharides through viscosity modifier and polymeric additive containing background electrolytes

The activation energy related to the electromigration of oligosaccharides can be determined from their measured electrophoretic mobilities at different temperatures. The effects of a viscosity modifier (ethylene glycol) and a polymeric additive (linear polyacrylamide) on the electrophoretic mobility of linear sugar oligomers with α1–4 linked glucose units (maltooligosaccharides) were studied in CE using the activation energy concept. The electrophoretic separations of 8‐aminopyrene‐1,3,6‐trisulfonate‐labeled maltooligosaccharides were monitored by LIF detection in the temperature range of 20–50°C, using either 0–60% ethylene glycol (viscosity modifier) or 0–3% linear polyacrylamide (polymeric additive) containing BGEs. Activation energy curves were constructed based on the slopes of the Arrhenius plots. With the use of linear polyacrylamide additive, solute size‐dependent activation energy variations were found for the maltooligosaccharides with polymerization degrees below and above maltoheptaose (DP 7), probably due to molecular conformation changes and possible matrix interaction effects.     

Boronate affinity saccharide electrophoresis: a novel carbohydrate analysis tool

The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore-assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5-1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar-containing molecules.     

Thin-layer electrophoretic separation of monosaccharides, oligosaccharides and related compounds on reverse phase silica gel

The electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols and sugar acids were determined in 0.3 M borate buffer, pH 10, using thin-layer electrophoresis on silanized silica gel, pretreated with octanol-1. A rapid separation of a number of sugars, occurring in foods, could be achieved. Using a 0.05-0.1 M neutral solution of barium acetate as electrolyte, thin-layer electrophoresis allowed excellent and rapid separation as well as identification of all common uronic acids which are constituents of many acidic polysaccharides.     

电中性分子间相互作用分析的毛细管电泳方法研究

使用具有负有效淌度的十二烷基磺酸钠胶束赋予中性分子有效淌度,以中性主体分子18-冠-6和中性客体分子对-硝基苯胺形成中性超分子包结化合物为实例,建立了中性分子相互作用的毛细管电泳分析方法,发现对-硝基苯胺和18-冠-6与SDS的相互作用存在相当的差异.测得18-冠-6和对-硝基苯胺的lgK为4.14,18-冠-6与2-氨基吡啶、18-冠-6与SDS以及SDS与2-氨基吡啶的lgK分别为3.65,3.13和1.15.     

Characterization of complexes between phenethylamine enantiomers and β‐cyclodextrin derivatives by capillary electrophoresis—Determination of binding constants and complex mobilities

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte‐cyclodextrin‐complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β‐cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x ‐reciprocal, y ‐reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β‐cyclodextrin, (2‐hydroxypropyl)‐β‐cyclodextrin, methyl‐β‐cyclodextrin and 6‐O‐α‐maltosyl‐β‐cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer‐cyclodextrin‐complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β‐cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes.     

Two-dimensional mapping by high-performance liquid chromatography of pyridylamino oligosaccharides from various glycosphingolipids.

A method to map sugars two-dimensionally for the analysis of the structures of oligosaccharides from glycosphingolipids is described. Nine neutral and ten acidic oligosaccharides were obtained from glycosphingolipids by endoglycoceramidase digestion and labelled with 2-aminopyridine. The pyridylamino oligosaccharides were clearly separated by high-performance liquid chromatography on commercially available C18-silica and amide-silica column. All compounds tested were mapped without any overlapping. The separation of the pyridylamino oligosaccharides on the C18-silica column depended on the numbers and positions of sialic acid and N-acetylhexosamine residues; on the amide-silica column, the separation depended on the total number of sugar residues.     

Determination of linkages of linear and branched oligosaccharides using closed-ring chromophore labeling and negative ion trap mass spectrometry

Linear as well as branched oligosaccharides were labeled with p-aminobenzoic ethyl ester (ABEE) using the glycosylamine closed-ring labeling approach and analyzed by negative-ion electrospray ionization mass spectrometry (ESI-MS). Linkage specific fragment ions of ABEE labeled linear oligosaccharides were proposed based on the MS2 and MS3 data for several ABEE labeled linear oligosaccharides with known linkage configurations. Fragmentation at the reducing end was similar to that observed for ABEE disaccharides whereas the fragmentation pattern not involving the reducing end was similar to underivatized disaccharides. Based on these ions, all the linkages of linear oligosaccharides could be unambiguously determined. The fragmentation pattern at the branched sugar was in general not quite the same as the linear one. However, many linkage specific fragment ions were also observed for linkages at the branched sugar. These ions along with the ions proposed for linear oligosaccharides were found to be quite useful for the determination of all the linkages of branched oligosaccharides.     

Effect of the matrix on DNA electrophoretic mobility

DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.     

Capillary zone electrophoresis of alpha 1-acid glycoprotein fragments from trypsin and endoglycosidase digestions

Capillary zone electrophoresis with fused-silica tubes having hydrophilic coating on the inner walls was evaluated in the separation of peptide and glycopeptide fragments from trypsin digestion of alpha 1-acid glycoprotein. Submapping of glycosylated and nonglycosylated tryptic fragments of the glycoprotein by capillary electrophoresis was facilitated by selective isolation of the glycopeptides on concanavalin A silica-based stationary phases prior to the electrophoretic run. In addition, the electrophoretic map and submaps of the whole tryptic digest and its concanavalin A fractions, respectively, allowed the elucidation of the microheterogeneity of the glycoprotein. Also, capillary zone electrophoresis proved suitable for the mapping of the oligosaccharide chains cleaved from the glycoproteins by endoglycosidase digestion. The oligosaccharides cleaved from human and bovine alpha 1-acid glycoprotein were analyzed after derivatization with 2-aminopyridine, which allowed their sensitive detection by on column UV absorption. The separation was best achieved when 0.1 M phosphate solution, pH 5.0, containing 50 mM tetrabutylammonium bromide was used as the running electrolyte. The effect of the organic salt on separation was attributed to ion-pair formation and/or hydrophobic interaction.     

Electrophoretic mobility of the polymer-like micelles of tetradecyldimethylamine oxide hemihydrochloride

Effects of the surfactant concentration C_d and the NaCl concentration C_s on the electrophoretic mobilities U of the well-characterized polymer-like micelles have been investigated by the electrophoretic light scattering, using tetradecyldimethylamine oxide hemihydrochloride (C14DMAO·1/2HCl). At the high ionic strength of 0.1 mol kg⁻¹ NaCl, the electrophoretic mobilities were independent of C_d (5 mM < C_d < 100 mM), despite the concentration-dependent micelle growth of the polymer-like micelles. This suggests that the electrophoretic mobility of the polymer-like micelle at high ionic strengths is independent of the contour length (i.e., the molecular weight), as found on linear polyelectrolytes. Somewhat surprisingly, the entanglements of the polymer-like micelles gave small effect on the electrophoretic mobilities in the examined range of the surfactant concentration above an overlap concentration. The mobilities of the polymer-like micelle decreased with √C_s in a single exponential manner in the range of C_s from 0.02 to 0.3 mol kg⁻¹. It is suggested that the cylinder model can be applied to the electrophoretic mobilities of the polymer-like micelles at high ionic strengths (i.e. a free-draining behavior), since the persistence length of the polymer-like micelle (20 nm) is much larger than the Debye length at high ionic strength.     

Determination of the ionization constants of weak electrolytes by capillary zone electrophoresis

Capillary zone electrophoresis in untreated fused silica capillaries has proved suitable for the determination of the ionization constants of weak electrolytes. Several fundamental equations relating the electrophoretic mobilities of ionized solutes to hydronium ion concentrations in the running electrolyte have been verified experimentally. The observed dependence of the electrophoretic mobilities of weak bases and ampholytes on the pH of the electrolyte showed good agreement with predicted behavior. The pK_a values calculated from electrophoretic mobility data obtained by capillary zone electrophoresis were reasonably close to those reported in the literature.     

Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

A hydrophilic polymer, poly(vinylpyrrolidone) (PVP), was employed for suppressing the electroosmotic flow (EOF). A capillary was filled with aqueous PVP solution for coating the capillary wall with PVP; the PVP solution was then replaced by a migration buffer solution containing no PVP. Three types of PVP with different molecular weights were examined. The EOF was suppressed more effectively as the molecular weight of PVP increased. The EOF in the coated capillary was approximately 10-fold smaller than that of a bare capillary and was constant in the pH range of 6-8. The suppressed EOF was stable even when no PVP was added to the migration buffer. However, the EOF increased significantly when sodium dodecyl sulfate was added into the migration buffer. The method was applied for determining the electrophoretic mobilities of inorganic anions that have negative electrophoretic mobilities larger than the electroosmotic mobility of the bare capillary. A novel method for determining the electrophoretic mobilities was proposed based on the linear relationship between electric current and electrophoretic mobility. The electrophoretic mobility was proportional to the electric current. Therefore, the intercept of the regression equation represents the electrophoretic mobility at room temperature. The electrophoretic mobilities were in good agreement with the absolute electrophoretic mobilities.     

Determination of molecular weight of native proteins by polyacrylamide gradient gel electrophoresis

An improved method for the estimation of molecular weights of native proteins by polyacrylamide gel electrophoresis, in 9 cm x 9 cm x 0.05 mm 4-20% T fabric reinforced gradient gels, is described. Plotting the logarithm of the relative mobilities of proteins versus gel concentrations produces lines whose slopes are related to molecular weights.     

Analysis of carbohydrates in glycoproteins by high-performance liquid chromatography and high-performance capillary electrophoresis

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE).O-andN-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection.N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.     

Determination of dissociation constants of amino acids by capillary zone electrophoresis.

A method is proposed for the determination of dissociation constants of amino acids by capillary zone electrophoresis. According to the dissociation equilibrium of amino acids and the basic theory of electrophoresis, the nonlinear relationship between the pH value of the buffer and the effective electrophoretic mobilities of the analyte was obtained. The dissociation constants can be calculated from the pH values and the corresponding effective electrophoretic mobilities using the program written in C++. The dissociation constants, pKa1 and pKa2, of 11 kinds of amino acids were determined successfully by the proposed method. The determined dissociation constants were compared with values in the literature; the differences between them are in the range of -0.03 to 0.06. No significant differences were observed between the determined dissociation constants and the corresponding literature values.     

Hydrodynamic suppression of the electroosmotic flow in capillary electrophoresis with indirect spectrophotometric detection

A version of capillary electrophoresis with indirect spectrophotometric detection and the hydrodynamic suppression of electroosmotic flow is studied. It is shown that, to improve the reliability of ion identification, one should calculate electrophoretic mobilities of ions or migration times corrected with regard to the electroosmotic flow rate. Correlations between electrophoretic peak areas of ions and their electrophoretic mobilities are derived. In the studied version of capillary electrophoresis, the accuracy of measuring anion concentrations can be improved using the internal standard method.     

Effect of monosaccharide composition, glycosidic linkage position and anomericity on the electrophoretic mobility of labeled oligosaccharides

Fluorophore-assisted carbohydrate electrophoresis (FACE) is useful for separation and characterization of oligosaccharides from various sources and for comparing several samples at once. While characterizing fungal surface glycans by FACE we observed that samples and standards of the same mass did not comigrate as expected. Subsequent experiments showed that the samples did not contain contaminating sugars. Therefore, our observation suggested that glycan electrophoretic mobility is affected by factors in addition to molecular mass. This work assesses the contribution of monosaccharide composition, linkage position, and linkage anomericity to glycan mobility. Commercially available (and synthesized when available) bioses of known composition were derivatized with a charged fluorophore, and electrophoretic mobilities compared in a slab gel format. The results indicate that all three parameters mentioned above affect observed migration. Further, no migration patterns emerged to suggest a set of rules for assigning band identity based on mobility alone. These results emphasize the importance of including known, matched, standards to facilitate interpretation of FACE data.     

Peptide mapping by capillary zone electrophoresis: how close is theoretical simulation to experimental determination

A multi-variable computer model is presented for the prediction of the electrophoretic mobilities of peptides at pH 2.5 from known physico-chemical constants of their amino acid residues. The model is empirical and does not claim any theoretical dependencies; however, the results suggest that, at least at this pH, peptides may be theoretically represented as classical polymers of freely joined amino acid residues of unequal sizes. The model assumes that the electrophoretic mobility can be represented by a product of three functions that return the contributions of peptide charge, length and width, respectively to the mobility. The model relies on accurate experimental determination of the electrophoretic mobilities of a diverse set of peptides, by capillary zone electrophoresis (CZE), at 22 degrees C, with a 50 mM phosphate buffer, at pH 2.5. The electrophoretic mobilities of a basis set of 102 peptides that varied in charge from 0.65 to 16 and in size from two to 42 amino acid residues were accurately measured at these fixed experimental conditions using a stable 10% linear polyacrylamide-coated column. Data from this basis set was used to derive the peptide charge, length, and width functions respectively. The main purpose of this endeavor is to use the model for the prediction of peptide mobilities at pH 2.5, and for simulation of CZE peptide maps of protein digests. Excellent agreement was obtained between predicted and experimental electrophoretic mobilities for all categories of peptides, including the highly charged and the hydrophobic. To illustrate the utility of this model in protein studies it was used to simulate theoretical peptide maps of the digests of glucagon and horse cytochrome c. The resulting maps were compared and contrasted with their experimental counterparts. The potential of this approach and its limitations are discussed.     

Capillary electrophoresis separation of vinpocetine and related compounds: prediction of electrophoretic mobilities in partly aqueous media

Offord's equation, a relationship between electrophoretic mobility and charge, size and shape of peptides, has been extended to quantitate the electrophoretic mobility of vinca alkaloids. Partly aqueous protonation constants and the derived theoretical mobilities have been proven to be able to predict experimental electrophoretic mobilities. In practice, seven vincamine derivatives of very low water-solubility were separated by capillary electrophoresis. Buffer total concentration, apparent pH and methanol content, the three most important parameters of the running buffer, were used in triangular resolution mapping to characterize separation. Even though electrophoresis is well known to slow down in partly aqueous media, under our optimized circumstances a baseline separation was achieved within 8 min in each case.