Microscale analysis of mucin-type O-glycans by a coordinated fluorophore-assisted carbohydrate electrophoresis and mass spectrometry approach

The total glycan moiety was released in a single step from native glycoproteins by a nonreductive beta-elimination procedure. The generated oligosaccharides were further derivatized either with the hydrophobic fluorophore 2-aminoacridone (AMAC) or the charged 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) fluorophore, and the resulting fluorescent derivatives were separated according to their hydrodynamic size or charge with high-resolution gel electrophoresis. Both N- and O-glycans released by this beta-elimination procedure might be analyzed simultaneously. AMAC derivatization allows a rapid separation of neutral and charged oligosaccharides without prior fractionation. Derivatized oligosaccharide species were then eluted from the gel slices and analyzed by mass spectrometry. This methodology allowed the rapid structural characterization of each glycan in term of monosaccharide composition and sequence. Using this technique we were able to screen several heterogeneous O-glycan mixtures isolated at the picomolar range from reference glycoproteins or mucins.     

Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence‐assisted carbohydrate electrophoresis

Fluorescence‐assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high‐throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β‐glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC 3.2.1.74. A hydrolysate study of a T. reesei Cel12A‐N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single‐site mutation on enzymatic activity.     

Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence‐assisted carbohydrate electrophoresis

The structure of xylan, which has a 1,4‐linked β‐xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence‐assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA²Xyl₃ and MeGlcA²Xyl₄, respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family‐specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β‐xylosidase degraded MeGlcA²Xyl₄ into xylose and MeGlcA²Xyl₃ but further degradation of MeGlcA²Xyl₃ required additional enzymes. Synergy between xylanases and β‐xylosidase was also found to significantly accelerate the conversion of XOS into xylose.     

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.     

Microchip extraction of catecholamines using a boronic acid functional affinity monolith

A novel solid phase extraction microchip with a boronic acid functional affinity monolithic disc was developed in this work. Vinyl phenylboronic acid–ethylene glycol dimethacrylate co-polymer monoliths, which have pore sizes up to 20 μm, were investigated for extraction of catecholamines using adsorption and desorption studies in a batch system. Desorption yields of greater than 90% were achieved for catecholamines at pH 3 and below. Monolithic discs were then formed in chambers in borofloat glass microfluidic chips using in situ UV polymerization. Adsorption on the monolithic discs was performed via electrokinetic flow, with catecholamines determined via laser-induced native fluorescence (LINF) detection following electrokinetic elution. Microchips containing the boronic acid functional polymer discs worked well for extraction of catecholamines, providing greater than 100 fold concentration enrichment. This study demonstrated that a solid phase extraction microchip, containing an easily prepared monolith disc, will be useful for boronate affinity extraction of cis-diol containing compounds.     

Pre-equilibrium capillary zone electrophoresis or frontal analysis: advantages of plateau peak conditions in affinity capillary electrophoresis

The feasibility of using the affinity CE methodologies pre-equilibrium CZE and CE frontal analysis was tested on interaction systems exhibiting rapid on-and-off kinetics. Experimentally, the methodologies differ only with respect to the volume of sample introduced into the capillary. Pre-equilibrium CZE has been considered amendable to interactions with slow on-and-off kinetics only; however, it has recently been applied in studies of interactions with fast on-and-off kinetics. The effect of varying the sample volume introduced hydrodynamically into the capillary on the apparent degree of complexation was studied. For two different binding systems, the fraction of free analyte was found to be overestimated using pre-equilibrium CZE as compared to volumes providing plateau peak conditions as used with frontal analysis. Results indicate that frontal analysis conditions lead to more robust binding assays and thus more reliable data. The validity of data obtained by pre-equilibrium CZE may be low, thus the use of an experimental setup providing plateau peaks is highly recommended. It is suggested that the effect of altering the sample volume on the degree of binding should be investigated as part of method development and validation.     

Cycling gradient capillary electrophoresis: a low-cost tool for high-throughput analysis of genetic variations

In the present work, we introduce a new type of DNA variation detection. This method represents a transfer of melting gel technique onto multicapillary electrophoresis DNA sequencing instrument with further improvements to achieve maximum sample throughput while maintaining a high performance. The main improvement comes from application of cycling (revolving) temporal temperature gradient in place of a single-sweep gradient, commonly used in similar gel-based techniques. This improvement enables utilization of multiple-injection technique, in which multiple samples are injected into the same capillary (or sets of capillaries) separated by predefined time intervals of partial electrophoresis. The periodic oscillation of the temperature results in identical separation conditions of all samples injected in such series. Using this novel approach, we demonstrate a dramatic increase in separation throughput by turning a standard commercial 96-capillary array instrument into a semicontinuous flow mutation detection system capable to screen over 15 000 samples in 24 h of operation on a single 96-capillary commercial instrument. This represents a 10-fold increase in sample throughput over the current comparable technology.     

Synthesis of a paramagnetic boronic acid as a useful synthetic building block and carbohydrate affinity spin probe

A paramagnetic boronic acid was synthesized by lithiation of 1-acetoxy-3-bromo-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole followed by treatment with trimethyl borate. This paramagnetic boronic acid proved to be a useful starting compound in Suzuki cross-coupling reactions and it exhibited affinity toward fructose and inulin.     

Determination of carbohydrates in Folium Lysium Chinensis using capillary electrophoresis combined with far-infrared light irradiation-assisted extraction

In this work, a method based on capillary electrophoresis with amperometric detection and far-infrared-assisted extraction has been developed for the determination of mannitol, sucrose, glucose and fructose in Folium Lysium Chinensis, a commonly used traditional Chinese medicine. The water-soluble constituents in the herbal drug were extracted with double distilled water with the assistance of far-infrared radiations. The effects of detection potential, irradiation time, and the voltage applied on the infrared generator were investigated to acquire the optimum analysis conditions. The detection electrode was a 300-μm-diameter copper disk electrode at a detection potential of +0.65 V. The four carbohydrates could be well separated within 18 min in a 50-cm length fused-silica capillary at a separation voltage of 9 kV in a 50-mM NaOH aqueous solution. The relation between peak current and analyte concentration was linear over about three orders of magnitude with detection limits (S/N=3) ranging from 0.66 to 1.15 μM for all analytes. The results indicated that far infrared significantly enhanced the extraction efficiency of the carbohydrates in Folium Lysium Chinensis. The extraction time was significantly reduced to 7 min compared with several hours for conventional hot solvent extraction.     

Two-dimensional electrophoresis protein profiling as an analytical tool for human acute leukemia classification

Two-dimensional electrophoresis (2-DE) was used to profile the proteins of leukemic cells from 61 cases of akute leukemia (AL) characterized by the French-American-British (FAB) classification. The differentially expressed protein spots were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). The distinct protein profiles (DPPs) of AL FAB subtypes were explored successfully, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5), and acute lymphoid leukemia (ALL), which were homogeneous within different samples of the same subgroup but clearly differed from all other subgroups. We also found a group of proteins differentially expressed between AL cells and normal white blood cells. Among the DPPs of AL subtypes, some proteins have been reported, but most of them were first reported here to mark AML differentiation and to discriminate AML from ALL. These data show that 2-DE protein profiling could be used as an analytical tool for facilitating molecular definition of human AL classification and understanding the mechanism of leukemogensis, and the extension of the present analysis to the currently less well-defined AL will identify additional subgroups and may promote the identification of new targets for specific treatment approaches.     

Further resolution of alpha-fetoprotein glycoforms by two-dimensional isoelectric focusing and lectin affinity electrophoresis

Human alpha-fetoprotein (AFP) from serum of patients with cirrhosis and hepatocellular carcinoma (HCC) was separated into several bands by IEF and by erythroagglutinating phytohemagglutinin (E-PHA) affinity electrophoresis. These AFP bands were directly compared in 2-D IEF and E-PHA affinity electrophoresis. IEF of serum AFP was run in 1% agarose IEF gel with 3% Pharmalyte 4.5-5.4. After IEF, a part of the gel was stained for AFP and another part of the gel corresponding to the area of separated AFP bands was cut in 1 mm x 39 mm along the focused direction and transferred to a trough in 1% agarose gel with 0.3 mg/mL E(4)-PHA for second-dimensional affinity electrophoresis. Separated 2-D AFP spots were visualized by antibody-affinity blotting and identified by combining the systems of Johnson et al.. (Johnson, P. J., Ho, S., Cheng, P., Chan, A. et al.., Cancer 1995, 75, 1663-1668) for AFP-I-+IV and of Taketa et al.. (Taketa, K., Ichikawa, E., Taga, H., Hirai, H., Electrophoresis 1985, 6, 492-497) for AFP-P1-5. AFP-P2, the major AFP glycoform, was composed of AFP-I, AFP+I, and AFP+II; AFP-P3, a nonspecific monosialo-AFP, was composed of AFP+II; AFP-P4, HCC-specific monosialo-AFP, was composed of AFP+II, AFP+III, and AFP+IV; and malignancy-related AFP-P5 was composed of AFP+I and AFP+II. Monosialo-AFP (AFP+II) was recovered in all the glycoforms of AFP-P2, -P3, -P4, and -P5; thus, AFP-P4 is more specific to HCC than monosialylated AFP+II.     

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 capillary electrophoresis for the assessment of binding affinity of carbohydrate‐based cholera toxin inhibitors

Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B‐subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1‐oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene‐dextran sulfate‐polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (K_d) determination. The K_d values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC₅₀ values of an ELISA‐type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen‐induced secretory diarrhea.     

Boronate functionalized magnetic nanoparticles and off-line hyphenation with capillary electrophoresis for specific extraction and analysis of biomolecules containing cis-diols

In recent years, functionalized magnetic nanoparticles (MNPs) have drawn continuously increasing attention due to their great potential for capturing biological molecules or species. However, functionalized MNPs as nanoextraction probes and the coupling with a separation platform for chemical analysis have not extensively investigated yet. In this study, boronate functionalized MNPs were synthesized and employed as extracting probes to capture and enrich cis-diol-containing biomolecules, and an off-line coupling method of the MNPs-based extraction with capillary electrophoresis (CE) was established by using pH junction, an on-line preconcentration technique in CE, as a bridge for the coupling. The prepared MNPs exhibited specific selectivity and sufficient capacity. The pH junction compressed a large injected sample volume into a much narrower sample zone and therefore significantly improved the detection sensitivity, solving the sensitivity mismatch between the MNPs-based extraction and CE. Experimental conditions for the pH junction and the desorption were optimized. Under the optimized conditions, the sensitivity was enhanced by 42-fold as compared with regular CE. N,N-dimethylformamide was found to be an effective desorption promoter, which reduced the desorption time to a few minutes. With the established method, riboflavin in a human urine sample was determined.     

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs

In this study, two capillary electrophoresis–based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l -tryptophan (l -TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l -TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.     

Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.     

Microalgae amino acid extraction and analysis at nanomolar level using electroporation and capillary electrophoresis with laser‐induced fluorescence detection

Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser‐induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave‐assisted derivatization at 80°C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and β‐cyclodextrin (10 mM), and the limits of quantification were 3–30 nM. The developed capillary electrophoresis with laser‐induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home‐made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated‐solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells.     

Enhancing separation of histidine from amino acids via free-flow affinity electrophoresis with gravity-induced uniform hydrodynamic flow

In this paper, a novel mode of free-flow affinity electrophoresis (FFAE) was developed to indirectly enhance the separation of free-flow electrophoresis (FFE). In the mode of FFAE, a Ni(II) with high electric charge density and histidine (His) is chosen as a model ligand and target solute, respectively. Through the controlling of experimental conditions (10 mM pH 6.0 Na(2)HPO(4)-NaH(2)PO(4) with 2.0 mM NiCl(2)·6H(2)O background buffer), Ni(II) can combine with His and the combination leads to the high electric charge density of affinity complex of His-Ni(II) in contrast to the low density of free His molecule. But the ligand has weak interaction with uninterested amino acids. Thus, the mobility of His existing as His-Ni(II) is greatly increased from 14.5×10(-8) m(2) V(-1) s(-1) to 30.2 × 10(-8) m(2) V(-1) s(-1), while those mobilities of uninterested amino acids are almost constant. By virtue of the mode, we developed the FFAE procedure and conducted the relevant experiments. The experiments demonstrated the following merits of the FFAE technique: (i) clear enhancement of separation between the target solute of His and uninterested amino acids; (ii) simplicity, and (iii) low cost. Furthermore, the technique was used for the continuous separation of His from its complex sample, and the purity of His was near to 100%. All of the results demonstrate the feasibility of affinity separation in FFE. The developed FFAE may be used in the separation and pretreatment of some biological molecules (e.g. peptides).     

On-line concentration of peptides and proteins with the hyphenation of polymer monolithic immobilized metal affinity chromatography and capillary electrophoresis

An iminodiacetic acid (IDA)-type adsorbent is prepared at the one end of a capillary by covalently bonding IDA to the monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate). Cu(II) is later introduced to the support via the interaction with IDA. By this means, polymer monolithic immobilized metal affinity chromatography (IMAC) materials are prepared. With such a column, IMAC for on-line concentration and capillary electrophoresis (CE) for the subsequent analysis are hyphenated for the analysis of peptides and proteins. The reproducibility of such a column has been proved good with relative standard deviations (RSDs) of dead time of less than 5% for injection-to-injection and 12% for column-to-column (n = 3). Through application on the analysis of standard peptides and real protein samples, such a technique has shown promising in proteome study.     

Validated method for L-ornithine-L-aspartate analysis in human plasma by capillary electrophoresis

A rapid capillary electrophoresis method for routine determination of two amino acids, L-ornithine and L-aspartic acid, in human plasma is reported. The method runs automatically, requires a minimum of sample preparation and moreover includes no extensive extraction and no gradient or derivatization procedure. Analyses were performed on an uncoated silica capillary using buffer solution composed with 10 mM sodium tetraborate and 1 M sodium hydroxide (pH=10.0). A capillary electrophoresis P/ACE system equipped with UV detection (200 nm), an automatic injector, a fluid cooled cartridge and System Gold data station was used in this study. The total analysis time under these conditions was 8.0 min. The calibration curve was linear in the range 10-280 microg mL-1 for L-aspartic acid and 20-280 microg mL-1 for L-ornithine (for both amino acids, r=0.999). The method was validated by inaccuracy (bias) and precision (RSD) studies by analysing samples. The method was successfully applied to the quantitative determination of L-ornithine-L-aspartate in human plasma and could be useful for clinical and bioavailability investigations.