Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.     

Capillary electromigration methods for the study of collagen

This review paper gives an overview of capillary electromigration methods used in the analysis of collagen. Analyses of the parent chains as well as of the bromcyane and collagenase fragments of collagens are presented. Methods include capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic chromatography as well as combinations of HPLC and capillary electrophoresis, and capillary electrophoresis with mass spectrometry.     

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.     

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.     

Affinity capillary electrophoresis: important application areas and some recent developments

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting (`plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen–antibody, hapten–antibody, lectin–sugar, drug–protein, and enzyme–substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.     

Enantioselective separations using capillary electrophoresis

The preconditions are outlined for enantioselective separations in capillary electrophoresis (CE) with chiral selectors as additives to the background electrolyte. Free solution capillary electrophoresis conditions are characterised by a single solution phase. Chiral separations are reviewed by selector type (chiral ligand exchange, cyclodextrins, crown ethers, glycoproteins) with the extensive studies on cyclodextrins grouped into sections on amino acids, pharmaceuticals, and speciality chemicals, optimisation, biological fluids, and quantitative aspects. In micellar electrokinetic capillary chromatography, enantioselective discrimination occurs by partition in a two-phase system, with a chiral micellar phase as selector. Optimum separation conditions can be readily predicted for a given selector–selectand combination, and absolute values of binding constants determined by CE. Advantages of CE in comparison with HPLC using a chiral stationary phase include robust, rapid assays and the use of small volumes of aqueous solutions; disadvantages include less favourable detection limits. © 1994 Wiley-Liss, Inc.     

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.     

Analysis of disease-causing genes and DNA-based drugs by capillary electrophoresis towards DNA diagnosis and gene therapy for human diseases

Rapid progress in the Human Genome Project has stimulated investigations for gene therapy and DNA diagnosis of human diseases through mutation or polymorphism analysis of disease-causing genes and has resulted in a new class of drugs, i.e., DNA-based drugs, including human gene, disease-causing gene, antisene DNA, DNA vaccine, triplex-forming oligonucleotide, protein-binding oligonucleotides, and ribozyme. The recent development of capillary electrophoresis technologies has facilitated the application of capillary electrophoresis to the analysis of DNA-based drugs and the detection of mutations and polymorphism on human genes towards DNA diagnosis and gene therapy for human diseases. In this article the present state of studies on the analysis of DNA-based drugs and disease-causing genes by capillary electrophoresis is reviewed. The paper gives an overview of recent progress in the Human Genome Project and the fundamental aspects of polymerase chain reaction-based technologies for the detection of mutations and polymorphism on human genes and capillary electrophoresis techniques. Attention is mainly paid to the application of capillary electrophoresis to polymerase chain reaction analysis, restriction fragment length polymorphism, single strand conformational polymorphism, variable number of tandem repeat, microsatellite analysis, hybridization technique, and monitoring of DNA-based drugs. Possible future trends are also discussed.     

Clinical applications of capillary electrophoresis

Capillary electrophoresis (CE) is an extremely sensitive technique, which has been used in the clinical laboratory for almost 10 yr. The components of CE instrumentation are described, as are injection modes, buffers, and effects of electroosmotic flow. The modes of separation used in CE, namely, capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic capillary chromatography, are explained. References for 26 different clinical applications of CE are included, among them assays that are used routinely as well as niche assays for specialized applications of CE. Verification of CE assays, current instrumentation, and future development of CE in the clinical laboratory are addressed.     

Capillary affinity gel electrophoresis: new technique for specific recognition of DNA sequence and the mutation detection on DNA

The present state of studies on capillary affinity gel electrophoresis, which is a new technique for the specific recognition of a target DNA sequence, is reviewed. This article includes the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the sequence-specific recognition of DNA and the detection of mutations in specific genes is illustrated.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Capillary electrophoresis of highly sulfated flavanoids and flavonoids

Flavanoids and flavonoids are natural products present in our diet and known to possess multiple biological activities. Sulfated species of these natural products represent highly charged water-soluble organic molecules that possess unique biochemical properties. We describe here the first studies on capillary electrophoresis of these highly charged molecules. Fully sulfated flavanoids and flavonoids can be electrophoresed and resolved under reverse polarity at pH 3.5 using 5-10 kV in less than 20 min. In contrast, at high pH under normal polarity these species can be electrophoresed only if a pressurized capillary is employed. (+/-)-Catechin sulfate, a racemic sulfated flavanoid, was resolved into its enantiomers using 15% beta-cyclodextrin, a chiral selector, but not with alpha- or gamma-cyclodextrins. Yet, the high charge density of these molecules challenges the resolving capability of capillary electrophoresis as diastereomers (-)-epicatechin sulfate and (+)-catechin sulfate do not resolve, even in the presence of cyclodextrins or chiral positively charged amino acids. Overall, capillary electrophoresis of highly sulfated flavanoids and flavonoids is expected to be useful in rapid structure analysis of sulfated flavonoids, either synthetic or natural.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Optimization of lambda-carrageenan as a chiral selector in capillary electrophoresis separations

Lambda-carrageenan, a linear high molecular weight sulfated polysaccharide, was employed as a chiral selector in capillary electrophoresis for the separation of enantiomers of weakly basic pharmaceutical compounds. In order to improve the utility of the chiral selector, the purity and concentration of the lambda-carrageenan and other important capillary electrophoresis method parameters were investigated. The results indicated that the purity and concentration of the lambda-carrageenan, ionic strength of the buffer, and temperature were critical to successful enantioseparation. These new method conditions were then applied to previously investigated beta-blockers (such as propranolol HCl and pindolol) and racemic tryptophan derivatives. These studies were successful in identifying important method conditions for the improved enantioselectivity with lambda-carrageenan.     

Capillary affinity gel electrophoresis

Capillary affinity gel electrophoresis is a new technique for the recognition of the specific DNA base and/or sequence. This technology is also applicable to the characterization of binding properties of DNA-based drugs, chiral separation, and the selective separation of antibody mimetics using imprinted polymers. This article reviews the present state of studies on the capillary affinity gel electrophoresis, including the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the detection of the mutation onDNA is illustrated.     

The use of capillary electrophoresis in a micropreparative mode: methods and applications.

The ability to collect sufficient quantities of analytes from capillary electrophoresis for subsequent analyses is demonstrated. Fractions collected have been analyzed using the following techniques: capillary electrophoresis, mass spectrometry, and protein sequencing. Fractions can be collected directly into small volumes of buffer or directly onto membrane surfaces. Relevant parameters such as capillary diameter, mass loading, and separation parameters are addressed.     

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE–mass spectrometry in forensic toxicology is finally given.     

Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices

Cells are the fundamental unit of life, and studies on cell contribute to reveal the mystery of life. However, since variability exists between individual cells even in the same kind of cells, increased emphasis has been put on the analysis of individual cells for getting better understanding on the organism functions. During the past two decades, various techniques have been developed for single-cell analysis. Capillary electrophoresis is an excellent technique for identifying and quantifying the contents of single cells. The microfluidic devices afford a versatile platform for single-cell analysis owing to their unique characteristics. This article provides a review on recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices; focus areas to be covered include sampling techniques, detection methods and main applications in capillary electrophoresis, and cell culture, cell manipulation, chemical cytometry and cellular physiology on microfluidic devices.     

Aggregation and denaturation of antibodies: a capillary electrophoresis, dynamic light scattering, and aqueous two-phase partitioning study

Protein denaturation and aggregation are well-known problems in the pharmaceutical industry. As the protein aggregates, it loses its biological activity and creates problems in its administration to patients. In this paper, we explore the use of aqueous two-phase systems, capillary zone electrophoresis, and dynamic light scattering for the monitoring of protein denaturation and aggregation. Our studies focus on human IgG and HSA. Capillary zone electrophoresis was used to monitor changes in the charge to size ratio of the proteins upon denaturation and dynamic light scattering was used to detect the presence of any aggregates and to monitor the size of the proteins. The information obtained from aqueous two-phase partitioning is similar to that obtained from capillary zone electrophoresis. The simplicity of aqueous two-phase system and its low cost (compared to the other analytical techniques) suggest that it can be routinely used for the quality control of some pharmaceutical preparations.     

On-chip capillary electrophoresis for alkaline phosphatase testing

We fabricated an on-chip capillary electrophoresis device for blood analysis. An on-chip capillary electrophoresis device was photolithographically fabricated on a glass chip. Alkaline phosphatase (ALP) was employed as a sample enzyme. Small amounts of enzyme in the mixture of other proteins were detected with the electrophoretically mediated microanalysis (EMMA) method. Fluorescein diphosphate was used as fluorogenic substrate. The detection of ALP activity was achieved with laser-induced fluorescence monitoring fluorescein that was produced in enzyme reaction in capillary. Several methods to reduce the adhesion of protein are also discussed.