High-performance capillary electrophoresis of cereal proteins

Cereal grains are widely used of human foods and animal feed throughout the world. Cereals provide dietary protein, which also often has a functional role, as wheat gluten does in bread. Cereal proteins are unique in many ways: they are highly complex and heterogeneous, are often difficult to extract, and aggregate readily, making them difficult to characterize. Because of the economic importance and widespread use of cereal proteins, however, many techniques have been used for their analysis. High-performance capillary electrophoresis (HPCE) is one of the newest techniques to be so used. This review describes the development of charge- and size-based HPCE methods for analysis of cereal grain proteins, and the use of these methods for cultivar identification, classification, and prediction of quality. HPCE is versatile, rapid, easily automated, readily quantified, and provides high-resolution separations. Clearly, HPCE is a valuable addition to other methods of cereal protein analysis and should, in time, be applicable to all protein classes from all cereals.     

Selectivity in capillary electrophoresis in the presence of micelles, chiral selectors and non-aqueous media

In addition to high efficiency, short analysis times and small sample volumes, a further attractive feature of capillary electrophoretic techniques is the possibility to achieve, high selectivities. Usually, selectivity control also allows improvement in the resolution. A simple way to enhance the selectivity of capillary electrophoretic separations is to add one or more surfactants above their critical micelle concentration, or in the case of chiral separations to add a chiral selector to the background electrolyte. Because of the dynamic structure of micelles, the aggregation of monomers and size of the micelles can be easily adjusted. This review describes the various type of surfactants used in micellar electrokinetic capillary chromatography, and the chiral selectors employed in enantiomeric separations by capillary electrophoresis. Factors affecting the selectivity are noted. A brief discussion is included of the selectivity enhancement obtainable in non-aqueous media.     

Purity testing of recombinant proteins by capillary electrophoresis

Purity testing of recombinant DNA (rDNA) proteins using slab gel electrophoresis in conjunction with scanning densitometry is time consuming and labor intensive and is difficult to reproduce because the dyes used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes a micellar capillary zone electrophoresis (MCZE) procedure that overcomes these difficulties. The MCZE method was evaluated to estimate protein purity of hydrophobic cytomegalovirus proteins, expressed E. coli, and highly glycosylated hepatitis C virus proteins, expressed in Chinese hamster ovary cells. The results obtained by the MCZE procedure correlated very well with the purity results quantitated by the conventional slab gel electrophoresis method using purified Coomassie Brilliant Blue dye to reduce anomalies. MCZE may serve as an alternative method for in-process and purity testing of rDNA proteins.     

Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.     

Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides

The complementary nature of anion exchange chromatography and capillary gel electrophoresis for oligonucleotide analysis is demonstrated by evaluating a comprehensive series of authentic deletion sequences and partial phosphodiester analogs of five phosphorothioate oligonucleotides of different base composition and sequence. While anion exchange HPLC is sensitive to differences in backbone length of phosphorothioate oligonucleotides, oligomers with length difference of one base unit are not resolved. Capillary gel electrophoresis, on the other hand, has excellent single-base resolution while being relatively insensitive to phosphate in the phosphorothioate backbone. The data definitively establish the necessity of employing both separation techniques for adequate characterization of lower order process-related impurities potentially found in synthetic phosphorothioate oligonucleotides.     

Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins

The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis. This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem. The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.2.1.17); (ii) a protein charge ladder obtained by acetylation of carbonic anhydrase II (EC 4.2.1.1); (iii) a test panel of proteins with a range of values of molecular weight and pI. Four polycationic polymers were examined: polyethylenimine (PEI; MWav = 15000), Polybrene (MWav = 25000), poly(methoxyethoxyethyl)ethylenimine (MWav = 64000), and poly(diallyldimethylammonium chloride) (MWav = 10000). Detection of proteins with high pI was readily achieved using the first three of these polycationic polymer coatings but not with the poly(diallyldimethyl-ammonium chloride). Examination of the stability of these coatings indicates that they are robust: the change in electroosmotic flow was less than 10% for 25 replications of the same separations, using capillaries coated with PEI or Polybrene. This study demonstrates that the charge ladder obtained by acetylation of lysozyme is a good model with which to test the efficiency of polycationic coatings. A study of the electrophoretic mobilities of the members of this charge ladder at pH 8.3 determined the effective charge of lysozyme (Zp(0) = +7.6 +/- 0.1) and established the acidity of the alpha-ammonium group of lysozyme (pKa = 7.8 +/- 0.1). Results from the test panel of proteins suggest that protein adsorption is mainly driven by electrostatic interactions.     

Clinical application of capillary electrophoresis to unconcentrated human urine proteins

Urine protein electrophoresis can be performed by capillary electrophoresis using spun unconcentrated urine diluted with running buffer. The separation which utilises the excellent sensitivity of capillary electrophoresis gives electropherograms similar to those obtained by diluting concentrated urine with buffer. A 72 cm x 50 microns internal diameter (ID) fused silica capillary was used for the analysis which took less than 15 min. Bence Jones protein can be detected from unconcentrated urine over urine protein concentrations ranging from 9.7 g/L to 0.04 g/L. Other clinical patterns, such as glomerular proteinuria, the presence of intact immunoglobulin and Tamm Horsfall protein can also be detected. The benefit of using unconcentrated urine is the time and cost saved by not concentrating the urine.     

Behavior and use of nonaqueous media without supporting electrolyte in capillary electrophoresis and capillary electrochromatography

Five nonaqueous solvents (acetonitrile, methanol, N,N-dimethylformamide, dimethyl sulfoxide, formamide) and deionized water were investigated for their ability to support electroosmotic flow (EOF) without electrolytic additives. In general, flow was found to be equal to or greater than flow with typical CE buffer systems. The magnitude of EOF was determined for each solvent by open tubular capillary electrophoresis (CE) and related to viscosity (eta), dielectric constant (epsilon), and the ratio of dielectric constant to viscosity (eta/epsilon). Zeta potentials (zeta) were derived indirectly from flow data and tabulated. Comparisons of flow behavior and zeta were made between pure solvents and conventional CE buffers, and questions of equilibrium and reproducibility were addressed. Similar experiments were performed using hydroorganic mobile phases (ACN/water, MeOH/water) across the complete compositional range (100% water-100% organic), with flow characteristics and zeta reported for each mobile phase system. Packed capillary columns (5-microns ODS) were evaluated for flow and retention stability under capillary electrochromatographic (CEC) conditions. A separation of 11 polycyclic aromatic hydrocarbons was performed in under 13 min by CEC with an ACN/water mobile phase. Reduced plate heights (h) were calculated between 2.5 and 3.0 for solutes with capacity factors (k') up to 4.5 for the most retained solute.     

Assessment of the quality of dairy products by capillary electrophoresis of milk proteins

This paper presents an overview of existing capillary electrophoretic methods for the study of milk proteins. The main methods of analysis of caseins, whey proteins and peptides are examined with particular attention to their application to the evaluation of the quality of dairy products. Aspects such as the study of protein polymorphism, evaluation of heat treatments, detection of adulteration and assessment of proteolysis are considered in detail.     

Capillary affinity chromatography and affinity capillary electrophoresis of heparin binding proteins

A new approach for separation, capillary affinity chromatography, is introduced for studying the interaction of heparin with antithrombin III and secretory leukocyte proteinase inhibitor. Heparin is covalently immobilized on the surface of an etched capillary through a silane spacer. The proteins are injected into the heparinized capillary, bound to the heparin, washed with buffer, eluted with sodium chloride in the same buffer using a pressure injection mode and eluting protein detected by absorbance. The resulting affinity separation is similar to that obtained from traditional affinity chromatography. The quantity of loaded protein in capillary affinity chromatography is at the nanogram level, offering an improvement over the milligram levels required for standard affinity chromatographic methods.     

Advances in capillary electrophoresis

This review summarizes the advancement in operational modes and selected applications of the title technique over the past five years. Regarding operational modes particular emphasis is put upon increasing selectivity and resolution, hyphenation of capillary electrophoresis with techniques based on other than electromigration principles, the so-called chip technology and new ways of detection. In applications selected examples of chiral separation and separation of biopolymers (proteins, nucleic acids) are emphasized. It is demonstrated that capillary electrophoresis represents a complementary technique to high-performance column chromatography and in a number of cases it offers better separations than standard chromatographic procedures.     

Validation of analytical capillary electrophoresis methods for use in a regulated environment

Since its introduction into the analytical laboratory, CE has had to prove that it was capable of generating results comparable to HPLC or GC techniques in the six areas (specificity, precision, accuracy, linearity, ruggedness, and range) typically required of validated methods intended for submission to governmental agencies. This paper will showcase the development and validation of two analytical methods: one for specific identification of HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethane sulfonic acid]) and the other to quantitate millimolar concentrations of tris (tris[hydroxymethyl]aminomethane) in high electrolyte solutions. Utilizing neutral markers and internal standards, results for HEPES demonstrate that migration time reproducibility, expressed as %RSD of 2% or less on a variety of capillaries, is obtainable. Additionally, for quantitation of tris, the values obtained for accuracy (%relative mean bias), precision (%RSD), and linearity (r2) over multiple days and capillaries meet the rigorous standards we require of HPLC or GC methods.     

Affinity capillary electrophoresis

The application of affinity capillary electrophoresis (ACE) to the study of molecular interactions is reviewed. ACE appears to be a sensitive, versatile and convenient tool to obtain reliable data on binding constants and stoichiometries of interacting systems using the Hummel-Dreyer method and variants thereof. A powerful feature is the possibility to analyze simultaneously the affinity of a large number of compounds for the same ligand, making it a promising tool for the screening of large combinatorial libraries.     

Two-dimensional separations: capillary electrophoresis coupled to channel gel electrophoresis

Two-dimensional separations provide extremely high peak capacities. Coupling capillary zone electrophoresis with ultrathin channel gel electrophoresis offers a convenient and efficient way to perform such two-dimensional microseparations. By means of in situ polymerization, high-concentration (up to 50%T) polyacrylamide gels are prepared in 75 mm long, 25 mm wide, and 40 microns thick rectangular channels. By moving the outlet end of the capillary electrophoresis capillary across the entrance of the channel, both separations are completely preserved. Mixtures of peptides labeled by fluorescein isothiocyanate (FITC) are well resolved in less than 15 min, with theoretical plate numbers in the range of 20,000-50,000 for each independent separation. Significant enhancement in separation efficiency and peak capacity over one-dimensional separations are demonstrated by this combination. The two-dimensional separations of a model mixture of peptides, a tryptic digest of trypsinogen, and < 0.05% of an individual B2 neuron from the marine mollusk Aplysia californica are presented.     

Column technology in capillary electrophoresis and capillary electrochromatography

A review of column technology in capillary electrophoresis (CE) including wall modification processes and open tubular as well as packed column formats in capillary electrochromatography (CEC) are presented. There are many approaches which can be used to solve separation problems which provide higher efficiency and/or shorter analysis times in comparison to other chromatographic techniques. However, both CE and CEC are still relatively undeveloped in comparison to a more mature method such as high performance liquid chromatography (HPLC) and improvement in column technology is changing rapidly.     

Determination of denatured serum proteins in the casein fraction of heat-treated milk by capillary zone electrophoresis

The amount of heat-denatured serum proteins in heat-treated milk could be estimated by analyzing the casein fraction, obtained by isoelectric precipitation at pH 4.6, by capillary zone electrophoresis. A hydrophilically coated capillary was used in combination with 6 M urea in a citrate buffer at pH.3. Optimization of the sample and running buffer minimized adsorption of serum proteins, especially that of bovine serum albumin (BSA). This afforded a detection limit down to ca. 5-65 micrograms/mL of the three main serum proteins in milk. The detector response (UV at 214 nm) was linear in the range of 0.05-0.35 and 0.05-0.85 mg/mL for alpha-lactalbumin and beta-lactoglobulin, respectively. BSA showed a slightly less linear behavior, due to residual adsorption to the capillary wall. The recovery of serum proteins was in the range of 89-107%. The method was evaluated by analyzing Dutch commercial milks and cheese milk, which had received increasing heat loads. The addition of milk powder to pasteurized milk could be detected by this method as well as the serum protein to casein ratio in various products.     

Chemiluminescence detection in capillary electrophoresis

Capillary electrophoresis (CE) and related techniques yield highly efficient separations while requiring only minute amounts of sample. Thus, these techniques are amenable to analyses of complex samples in diverse matrices and in situations where sample is extremely limited. The constraints of on-column detection generally result in poor detection limits and have reduced the overall application of CE. One logical approach to increased sensitivity in CE detection has been the development of chemiluminescence (CL)-based detectors. The current state of post-column detector development, CL applications, and limitations of the technique are reported herein.     

Trace analysis of proteins by capillary zone electrophoresis with on-column transient isotachophoretic preconcentration

The qualitative and quantitative aspects of transient isotachophoretic (ITP) sample preconcentration in the capillary zone electrophoretic analysis of protein samples have been demonstrated. By the proper selection of components of the background electrolyte and/or additives to the sample solution, two basic electrolyte arrangements have been employed. In the first, a typical isotachophoretic electrolyte system consisting of a leading and terminating electrolyte was used, and after focusing and preconcentration, the terminating electrolyte was replaced by the leading electrolyte, with the separation being continued in the zone electrophoretic mode. In the second, only one background electrolyte was used, containing a co-ion with low electrophoretic mobility, and the sample was supplemented with a salt of a highly mobile co-ion. In this case transient isotachophoretic migration of the sample ions took place at the beginning of the migration and gradually changed to the zone electrophoretic mode. Sample mixtures containing basic (positively charged) or acidic (negatively charged) proteins were examined using surface-coated fused-silica capillaries. For acidic proteins, bare silica was also tested. The isotachophoretic sample stacking permitted injection and preconcentration of sample volumes two to three orders of magnitude higher than usual in capillary zone electrophoresis. For example, up to 1 microL was injected into a 75 microns ID capillary. This approach afforded quantitative analysis of protein samples in the concentration range of 10(-7)-10(-8) M, with detection limits of approximately 10(-9) M. Furthermore, with constant sample volume injected, good reproducibility of migration times was obtained. Finally, the determination of trace components in the presence of a major sample component using transient ITP preconcentration has been demonstrated.     

Synergism of capillary isotachophoresis and capillary zone electrophoresis

Commercially pure titanium (CPT) substrate was subjected to porcelain firing and bond strengths under three-point bending mode (span length: 15 mm; crosshead speed: 0.5 mm/min) were evaluated. Experimental variables included surface treatments of CPT and porcelain firing schedules. Variables for the surface treatments were (1) sandblasting, (2) mono- and triple-layered nitridation, and (3) mono-layered chrome-doped nitridation. Variables for the porcelain firing schedule included (4) bonding agent application, (5) bonding agent plus gold bonding agent application, and (6) Procera porcelain application. All together eleven sample groups were prepared with different combination of aforementioned experimental variables. Statistically all of them exhibited no significant differences. Hence, we employed two further criteria; (I) the minimum bond strength should exceed the maximum porcelain strength per se, and (II) the CPT substrate should not be heated close to the beta-transus temperature. After applying these criteria, it was concluded that mono-layered nitridation and mono-layered application of chrome-doped nitridation on both (with and without) sandblasted and non-sandblasted surfaces were the most promising conditions for a successful Titanium-Porcelain System.     

Detection of carbohydrates in capillary electrophoresis

This review focuses on recent developments in sensitive detection modes for carbohydrates after separation by capillary electrophoretic methods. To bring detection sensitivity for carbohydrates analysis in line with current methods in protein sequencing, concentration detection limits of 10(-6) molar or better are required. A discussion of mass detection limits and concentration detection limits is followed by an overview of detection modes for natural and labeled carbohydrates. Amperometric detection and UV and laser-induced fluorescence detection after reductive amination, in particular with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), are discussed in more detail. Finally, the paper outlines developments to be expected in the near future, focusing on the needs in glycobiology such as improved sensitivity and selectivity.