Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Dependence of electroosmotic flow in capillary electrophoresis on Group I and II metal ions

The effect of Group I and II metal ions on electroosmotic flow in capillary electrophoresis in fused-silica capillaries is characterized. The electroosmotic mobility of aqueous mobile phases of lithium, sodium, potassium, calcium and barium acetates in fused-silica capillaries is measured as a function of pH at constant voltage. Cross contamination is avoided by using separate columns for each study and pH control is maintained with the aid of He sparging. The shape of a plot of pH vs. electroosmotic mobility depends on the particular cation used which in turn depends on the surface sorption properties of the ions. Column history is demonstrated to have an effect on electroosmotic flow and therefore retention times. The resolution of a test mixture is optimal in the lithium-based buffer.     

High-performance separation of polynucleotides by capillary gel electrophoresis.

Capillary gel electrophoresis was applied to the high speed separation of DNA and RNA. Factors affecting resolution and speed were optimized for the single base resolution of polynucleotides. Polynucleotides up to 350 bases were completely resolved within 38 min under optimum conditions.     

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.     

Screening method for inherited disorders of purine and pyrimidine metabolism by capillary electrophoresis with reversed electroosmotic flow

Capillary electrophoresis with electroosmotic flow reversed by cationic surfactant for diagnosis of purine and pyrimidine inherited enzyme deficiencies is reported. Final separation conditions consist of 45 mM borate, 55 mM N-tris[hydroxymethyl]methylglycine, 10 mM tartrate, 1 mM cetyltrimethylammonium bromide and 0.44% tetrabutylammonium hydroxide-2-amino-2-methyl-1,3-propanediol (pH 8.6). Average sensitivity (2.51 microM), reproducibility of migration times (run-to-run C.V. < or = 0.6%, day-to-day C.V. < or = 2.5%), linearity (R2>0.994) and imprecision (mean intra-assay RSD 4.7% and inter-assay RSD 6.6%) of the method are acceptable for diagnostic purposes. Applicability of the method is demonstrated on urine samples from patients with enzymatically proven enzyme deficiencies.     

High-performance capillary electrophoresis measurement of dolastatin-10

A high-performance capillary electrophoresis (HPCE) assay was used to determine the concentration of a potent cytotoxic agent, dolastatin-10, in human plasma. Following extraction from plasma, using a solid-phase C₁₈ cartridge, capillary zone electrophoresis was used to separate, detect and quantitate dolastatin-10 using the structurally related compound dolastatin-15 as the internal standard. Migration times for both dolastatins are less than 20 min. The recovery of the drug was approximately 90% and was quantified over the assay range of 39 to 5000 ng/ml with good precision and accuracy. The method is linear up to 5000 ng/ml with a lower limit of detection of 25 ng/ml. Data resulting from the use of the assay for the in vitro metabolism of the drug are presented. This is the first report of a validated HPCE assay for determining dolastatin-10 levels in human plasma.     

High-performance capillary electrophoresis: Protein charge estimations and peptide mapping by complexation electrophoresis

Two high-performance capillary electrophoretic (HPCE) methods are presented: The first methodology provides a procedure for estimating the isoelectric points of proteins in the absence of chaotropic agents with charge reversal Micro-Coat capillaries. The second method provides an optimized peptide mapping methodology for protein characterization that employs ion-pairing reagents to optimize the HPCE separation. Advantages and limitations of each methodology are discussed in terms of theory and practical experience. Both methodologies are applicable to a variety of proteins and both enhance our ability to characterize proteins on a molecular level.     

High-performance capillary electrophoresis of sialylated oligosaccharides of human milk

Oligosaccharides in human milk inhibit enteric pathogens in vitro and in vivo. Neutral milk oligosaccharides vary among individuals and over the course of lactation. To study such variation in the acidic milk oligosaccharides, a sensitive, convenient, quantitative method is needed. High-performance capillary electrophoresis of underivatized acidic oligosaccharides with detection by UV absorbance at 205 nm proved to be sensitive to the femtomole level. Eleven standard oligosaccharides ranging from tri- to nonasaccharide (3'-sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'-sialyllactosamine, disialyltetraose, 3'-sialyl-3-fucosyllactose, sialyllacto-N-tetraose-a, sialyllacto-N-tetraose-b, sialyllacto-N-neotetraose-c, disialyllacto-N-tetraose, and disialomonofucosyllacto-N-neohexaose) were resolved; baseline resolutions of 3'-sialyllactose, 6'-sialyllactose, and other structural isomers were achieved. Peak areas were linear from 30 to 2000 pg and were reproducible with a coefficient of variation between 4 and 9%. There was no evidence of quantitative interference of one oligosaccharide with another. In studies using pooled human milk, addition of increasing amounts of authentic standard oligosaccharides produced the expected positive increments in detected values, indicating quantitative recovery without interference by other milk components. The identities of the major sialylated acidic oligosaccharides of pooled human milk agreed with the results of previous studies employing other analytical methods. Comparison of oligosaccharide profiles of milk samples from different donors revealed extensive variation, especially in the structural isomers of sialyllacto-N-tetraose. This sensitive, highly reproducible method requires only simple sample workup and is useful in defining variations in human milk acidic oligosaccharides and investigating their possible relationship with diseases of infants.     

Microchip capillary gel electrophoresis of multiply PEGylated high-molecular-mass glycoproteins

PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation ("MCGE fingerprinting") of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range.     

High-performance capillary electrophoresis for determining HIV-1 Tat protein in neurons

The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders.     

Application of high-performance capillary electrophoresis to analysis of carbohydrates, especially those in glycoproteins

Methods for the component monosaccharide analysis and oligosaccharide mapping for glycoprotein research, based on HPCE of reductively pyridylaminated (PA) derivatives, are described. the component monosaccharides released from glycoproteins by acid hydrolysis are converted to PA derivatives and analyzed by HPCE as borate complexes. They can be quantified in the picomole range (introduced amount) with high reproducibility. The oligosaccharides released by hydrazinolysis are similarly converted to PA derivatives. Two-dimensional mapping of the relative mobilities of these derivatives, obtained in an acidic phosphate buffer and an alkaline borate buffer, ensures reliable identification of the oligosaccharides.     

HiTRACE: high-throughput robust analysis for capillary electrophoresis

MOTIVATION: Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment. RESULTS: We propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. We illustrate the application of HiTRACE on 13 datasets representing 4 different RNAs, 3 chemical modification strategies and up to 480 single mutant variants; the largest datasets each include 87 360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate datasets. Furthermore, while the smallest of these datasets required 7-10 h of manual intervention using prior approaches, HiTRACE quantitation of even the largest datasets herein was achieved in 3-12 min. The HiTRACE method, therefore, resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands.     

Fluorescence correlation spectroscopy for ultrasensitive DNA analysis in continuous flow capillary electrophoresis

Continuous flow capillary electrophoresis (CFCE) is non-separations based analytical technique based on the free solution electrophoretic mobility of biological molecules such as DNA, RNA, peptides, and proteins. The electrophoretic mobilities and translational diffusion constants of the analyte molecules are determined using single molecule detection methods, including fluorescence correlation spectroscopy (FCS). CFCE is used to resolve multiple components in a mixture of analytes, measure electrophoretic mobility shifts due to binding interactions, and study the hydrodynamic and electrostatic properties of biological molecules in solution. Often this information is obtained with greater speed and sensitivity than conventational separations-based capillary-zone electrophoresis. This paper will focus on the application of two-beam fluorescence cross-correlation spectroscopy as a versatile detection method for CFCE and explore several applications to the study of the solution properties of single-stranded DNA.     

Use of organic modifiers to enhance chiral selectivity in capillary electrophoresis

Summary Using capillary electrophoresis, the enantiomers of a number of dansyl amino acids were resolved using native-cyclodextrin. The neutral chiral host resolved analytes possessing a negative charge at pH 9, the conditions employed in this study. Organic modifiers added to the running buffer were particularly adept at enhancing chiral recognition between the guest and host molecule in capillary electrophoresis. This work examined the effects of methanol, dimethylformamide, and acetonitrile on the resolution, migration time, and efficiency of twelve dansyl amino acids. Examples are given of the separation of racemic dansyl amino acids utilizing this technique and conditions necessary to achieve enantioselectivity.     

Analysis of the microheterogeneity of the IgA1 hinge glycopeptide having multiple O-linked oligosaccharides by capillary electrophoresis

It was found that the self-aggregation of IgA1 was closely connected with the glycoform of a mucin-type sugar chain on its hinge portion. In this report, normal human serum IgA1 was separated into two subfractions by a jacalin column. The elution condition, 25 mM galactose, used here was similar to that reported for the glycoprotein with a single mucin-type sugar chain per molecule. The IgA1 eluted under this condition was substantially the monomeric form. In contrast, the remaining IgA1 eluted from the column with 0. 8 M galactose was substantially the aggregated form. An analytical method for the microheterogeneity of the IgA1 hinge glycopeptide (HGP33) was developed to determine the difference between these IgA1 fractions by capillary electrophoresis (CE). Native HGP33 from both IgA1 fractions was separated into peaks 1-11, depending on their glycoforms. Because the sialic acid-rich component migrated slowly on CE, the 25 mM fraction was abundant in the sialic acid-rich components (peaks 7-11), but the 0.8 M fraction was abundant in the sialic acid-poor components (peaks 1-4). Comparison of the number of sugar chains per hinge peptide indicated that the 25 mM fraction was relatively well glycosylated. Thus, application of CE analysis to the HGP33 indicated that the monomeric IgA1 was composed of a relatively complete molecule with respect to the glycoform rather than the aggregated IgA1.     

DNA analysis by microfabricated capillary electrophoresis device.

The LIGA (Lithographie Galvanoformung Abformung) process using synchrotron radiation lithography is applied to the microfabrication of capillary array electrophoresis (CAE) device. Laser-induced fluorescence detection system for the CAE device has been constructed by the modification of laser confocal fluorescence microscopy. DNA molecules were detected during migrating in the microchannels filled with polymer separation matrices under electric field to optimize the separation conditions for DNA analysis. Based on this observation, we demonstrated that microfabricated CAE device is realized the fast separation of DNA.     

High-performance capillary electrophoresis analysis of mate infusions prepared from stems and leaves of Ilex paraguariensis using automated micellar electrokinetic capillary chromatography

An automated micellar electrokinetic capillary chromatographic method has been developed in order to determine xanthines, e.g. caffeine, theobromine and theophylline, and chlorogenic acid in samples of yerba mate (Ilex paraguariensis). The target constituents were detected by photodiode array, and quantified by an external standard method. In addition, each constituent was collected separately and identified by EIMS. The method has been used to analyse 30 samples of mate infusions prepared at 30 and 75 degrees C with milled leaves and stems of 14 commercial brands which had been subjected to different elaboration processes. Suspended powdered material of each infusion was also analysed after three sieving steps. There was a remarkable difference in the relative xanthine composition of the finely suspended material, the amount of which varied according to the yerba mate brand, the elaboration process and the temperature of the infusion. The importance of these results with respect to gastrointestinal disorders which have been observed by habitual consumers of mate are discussed.     

High-performance anion-exchange chromatography with pulsed amperometric detection for carbohydrate analysis of glycoproteins

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an established technique for the carbohydrate analysis of glycoproteins. HPAE-PAD is routinely used for determinations of monosaccharide, sialic acid, mannose-6-phosphate (M-6-P), and oligosaccharide contents of a glycoprotein. This is true for both the initial investigation of a glycoprotein and routine assays of recombinant therapeutic glycoproteins. This contribution reviews the fundamentals of HPAE-PAD, recent technological improvements, and advances in the last ten years in its application to carbohydrate analysis of glycoproteins. The application areas reviewed include monosaccharide determinations, sialic acid determinations, M-6-P determinations, sugar alcohol determinations, analysis of polysialic acids, neutral and charged oligosaccharide analysis, following glycosidase and glycosyltransferase reactions, and coupling HPAE-PAD to mass spectrometry (MS).