Staining method for protein analysis by capillary gel electrophoresis

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS-protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS-protein-dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS-protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli.     

Determination of the compositional distribution of copolymers by frontal analysis continuous capillary electrophoresis

Frontal analysis continuous capillary electrophoresis (FACCE) was carried out for a series of random copolymers of an ionic monomer, sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS), and a nonionic monomer, acrylamide (AAM). The electropherograms appeared in order of anionic content and were generally sigmoidal, in contrast to that of hyaluronic acid (HA), which was abrupt and discontinuous. This difference could be related to the compositional heterogeneity of the copolymers, completely absent in the biopolymer. Through the range of copolymer composition (10-100% AMPS) the relationship between average mobility and nominal AMPS content could be described by a calibration curve, making it possible to deduce the compositional distribution of copolymer samples.     

Pressure-based approach for the analysis of protein adsorption in capillary electrophoresis

Protein adsorption to inner capillary walls creates a major obstacle in all applications of capillary electrophoresis involving protein samples. The problem is especially severe in kinetic capillary electrophoresis (KCE) techniques, which are used to study protein-ligand interactions at physiological conditions and, thus, cannot utilize extreme pH. A variety of coatings exist to reduce protein adsorption in CE, each expressing a unique surface chemistry that interacts with individual proteins differently. Here we introduce a simple pressure-based method for the qualitative assessment of protein adsorption that can facilitate the direct antiadhesive ranking of several coatings toward a protein of interest. In this approach, a short plug of the protein is injected into a capillary and propagated through with a pressure low enough to ensure adequate Taylor dispersion. The experiment is performed with a nonmodified commercial instrument in a pseudo-two-detector approach. The two detectors are mimicked by using two different distances from the capillary inlet to a single detector. If the peak area and shape do not change with changing distance, the protein does not adsorb appreciably, while a decreasing peak area with increasing distance infers inner surface adsorption. The magnitude change of the peak area between the two distances along with the overall peak shape is used to gauge the extent of protein adsorption. By using this method, we ranked antiadhesive properties of different wall chemistries for a series of proteins. The described method will be useful for optimizing protein analysis by CE and, in particular, for KCE experiments that investigate how proteins interact with their respective ligands.     

Determination and modeling of peptide pKa by capillary zone electrophoresis

In this work, pKa values of polyglycines, poly(L-alanines), and poly(L-valines) with a number of residues up to 10 were determined in different conditions of ionic strength (10 and 100 mM) and temperature (from 15 to 60 degrees C) by capillary electrophoresis. For each peptide family, the pKa values were modeled as a function of the number of residues, the temperature, and the ionic strength. Next, using this set of experimental data, a semiempirical model was developed in order to predict pKa values for any oligopeptide having neutral lateral chains. This model only needs, as input parameters, the number of residues and the pKa of terminal amino acids in their free form. It can predict the peptide pKa values at a given ionic strength and temperature. Comparisons with experimental data from the literature demonstrated that the prediction was possible with a standard deviation of approximately 0.1 pH unit.     

Quantitative analysis of beta-blockers in pharmaceutical preparations by capillary electrophoresis

A simple capillary electrophoresis method was developed for the analysis of four β-blockers (atenolol, metoprolol, pindolol, and propranolol) in pharmaceutical preparations. The method was validated regarding accuracy, precision, linearity, and detection/quantification limits, and the obtained values were in accordance to those reported in the literature. The method was applied to the determination of the drugs in commercial tablet preparations and proved to be fast and reliable for the quantitative analysis of the β-blockers.     

Analysis by capillary electrophoresis of the kinetics of charge ladder formation for bovine carbonic anhydrase

A series of charge ladders of bovine carbonic anhydrase II were synthesized and the relative abundances of the rungs analyzed by capillary electrophoresis as a function of the quantity of acylating agent used. A simulation that models the kinetics of formation of the members of the charge ladders is described. The observed rate constants decreased as the extent of acylation increased. These rate constants correlated adequately with theoretical rate constants calculated using Debye-Hückel theory. The data are compatible with, but do not demand, a model for the formation of this charge ladder in which all unacetylated amino groups in each rung have indistinguishable reactivity and in which the reactivity of the amines in each rung decreases as the net charge on the protein increases; in this model, decreased reactivity is due to increased extent of protonation. This agreement between experiment and model suggests that the charge shielding that results from an ionic strength of 130 mM is not sufficient to suppress the influence of the increasingly negative charge of the protein with acetylation on the extent of protonation of Lys epsilon-NH2 groups.     

Mobility moment analysis of molecular interactions by capillary electrophoresis

A new method for the quantitative evaluation of molecular interactions that are observed in electrophoresis is described. One component taking part in the interaction is labeled with a fluorescent dye and is subjected to capillary zone electrophoresis with fluorescence detection in the presence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant time intervals, and the electropherograms are converted to represent the fluorescence signal against mobility. After baseline subtraction, the first statistical moment of fluorescence signals on the mobility axis is calculated. This moment represents the average mobility of a labeled component. The change in the mobility moment in the presence and absence of the unlabeled component is used to evaluate the degree of saturation of the binding site of a labeled molecule with an unlabeled molecule. Mixtures of fluorescence-labeled protein (Fab' fragment of antibody or concanavalin A) and its unlabeled interacting partner (alpha(1)-antitrypsin or succinylated ovalbumin, respectively) at various concentrations were injected into a bare-silica capillary, and zone electrophoresis was carried out. The change in the mobility moment of the fluorescence-labeled molecules was used to determine the dissociation constants of the complexes. The determined constants are comparable to those obtained by a well-established method, that is, an analysis based on the peak height of the complex. Since the mobility moment analysis is not affected by the total intensity of the signals, it should be advantageous in analyses in which multiple capillaries are used, in which the injection volume and the sensitivity of detection might be more difficult to control at constant values. The mobility moment analysis also has advantages for the analysis of heterogeneous samples, since the identification of peaks is not necessarily required.     

Determination of nucleotides in fish tissues using capillary electrophoresis

Capillary electrophoresis has been applied to quantitate nucleotide degradation in fish tissues, to provide a basis for determining the K value, an indicator of fish freshness. The three major compounds, inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx) were distinctively separated at 416 V/cm applied potential, 100 mM CAPS buffer, pH 11. There was a good correlation between the peak area and the nucleotide concentration. By using a short distance (22 cm) from the sample entrance to the detector, the identification and determination of these compounds in each sample were completed within 15 min. The results obtained correlated very well with those obtained by enzymatic assays. The capillary was completely regenerated with 1 N NaOH, to dissociate all bound materials from the capillary wall, mainly cations in the fish extract. This provided the same silica surface for repeated runs, resulting in reproducible electropherograms.     

Field amplified separation in capillary electrophoresis: a capillary electrophoresis mode

In field-amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electroosmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field-amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field-amplified zone, using what we have called field amplified separation in capillary electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near-zero eof at the selected separation pH. Using FAsCE, improvements of 20% for separation speed and 40% for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, a part of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient, and fast CE separation mode.     

Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis

A method for determining the accurate effective mobility value of an analyte in the presence of a charged interacting agent, such as a charged cyclodextrin, a micellar agent, a protein, or a DNA fragment that binds the traditional electroosmotic flow markers, is presented. Part of the capillary is filled with the charged interacting agent-containing background electrolyte; the other part is filled with the charged interacting agent-free background electrolyte. The analyte band is placed in the charged interacting agent-containing background electrolyte zone, while a neutral marker (electroosmotic flow marker) is placed in the adjacent charged interacting agent-free background electrolyte zone. The initial, preelectrophoresis distance between the analyte band and the neutral marker band is determined by pressure mobilizing the bands past the detector and recording the detector trace. Subsequently, by applying reverse pressure, the bands are moved back into the first portion of the capillary and a brief electrophoretic separation is carried out. Then, the bands are pressure mobilized again past the detector to obtain their final, postelectrophoresis distance. If (i) the neutral marker does not come into contact with the charged interacting agent and (ii) the analyte does not migrate out of the homogeneous portion of the charged interacting agent zone, the accurate effective electrophoretic migration distance of the analyte, corrected for bulk flow transport, can be determined. The actual electric field strengths in the different zones of the heterogeneously filled capillary can be calculated from the integral of the electrophoretic current and the conductivity of the charged interacting agent-containing background electrolyte measured in a separate experiment. Once the effective mobility of an analyte in the charged resolving agent-containing background electrolyte is determined by this method, the analyte becomes a mobility reference probe for that background electrolyte and can be used to calculate the bulk flow mobility in subsequent conventional CE separations utilizing the same charged interacting agent. The new method can also be used to probe the interactions of the charged interacting agents and the wall of the capillary.     

Pteridine analysis in urine by capillary electrophoresis using laser-induced fluorescence detection

Pteridines are a class of compounds excreted in urine, the levels of which are found to elevate significantly in tumor-related diseases. For the first time, we have developed a method, based on high-performance capillary electrophoresis (HPCE) and laser-induced fluorescence (LIF) detection, to monitor the pteridine levels in urine. HPCE provides better separation than high-performance liquid chromatography and the LIF detector enables us to detect minute amounts of pteridines in body fluid. Eight different pteridine derivatives were well separated in 0.1 M Tris-0.1 M borate-2 mM EDTA buffer (pH 8.75) using a 60-cm fused-silica capillary (50-micron i.d., 35-cm effective length), six of which were detected and characterized in urine samples from normal persons and different cancer patients. The detection limits of these pteridines are under 1 x 10(-10) M. The levels of neopterin, pterine, xanthopterin, and pterin-6-carboxylic acid were found to be significantly elevated in urine excreted by cancer patents, while the level of isoxanthopterin dropped in these patients. No significant change of biopterin level was found between healthy individuals and cancer patients. This method can be used in clinical laboratories either for cancer monitoring or for precancer screening.     

Size-based characterization by the coupling of capillary electrophoresis to Taylor dispersion analysis

In this work, a new methodology is presented for performing capillary electrophoresis (CE) coupled to Taylor dispersion analysis (TDA). The CE step allows the separation of the different compounds of the injected mixture, while the diffusion coefficient related to each sample zone can be derived from the subsequent TDA step. TDA is an absolute and straightforward nonseparative method allowing the determination of the diffusion coefficient (or hydrodynamic radius) from the peak dispersion obtained in an open tube under Poiseuille laminar flow conditions. With a mass concentration sensitive detector, the hydrodynamic radius derived from TDA is a weight average value calculated upon all the molecules present in the sample zone. Since CE can be hardly coupled to light scattering detection for technical reasons (low volumes, short detection path length), TDA represents an interesting alternative for the size characterization, without calibration, of sample mixtures using CE-based separation techniques. The coupling of CE to TDA can be implemented on a commercial CE apparatus.     

Continuous cell introduction for the analysis of individual cells by capillary electrophoresis

Instrumentation for high-throughput analysis of single cells by capillary electrophoresis is described. A flow-based interface that uses electroosmotic flow (EOF) provides continuous injection of intact cells through an introduction capillary into a cell lysis junction and migration of the resulting cell lysate through a separation capillary for analysis. Specifically, two capillaries were coupled together with 5-mm-long Teflon tubing to create a approximately 5-microm gap, and the junction was immersed in a buffer reservoir. High voltage was applied across both capillaries so that cells were continuously pumped into the first capillary by EOF. Individual cells were lysed on-column at the junction without detergents, presumably owing to mechanical disruption caused by a dramatic change in flow properties at the gap. After each cell was lysed at the junction, the major proteins hemoglobin and carbonic anhydrase were separated by capillary electrophoresis and the resultant analyte zones were detected by laser-induced native fluorescence using 275-nm excitation. The detection limits of hemoglobin and carbonic anhydrase were 37 and 1.6 amol, respectively, which correlate well with the literature. The instrumentation was evaluated with intact red blood cells. The averaged time for complete analysis (i.e., continuous injection, lysis, separation, and detection) of one human erythrocyte was less than 4 min with this capillary-based setup. Moreover, this instrumentation simplifies the introduction of individual, intact cells without the use of a microscope.     

High resolution for single-strand conformation polymorphism analysis by capillary electrophoresis

Since the successful completion of the Human Genome Project, increasing concern is being directed toward the polymorphic aspect of the genome and its clinical relevance. A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab-gel electrophoresis (SGE) is applicable to the genetic diagnosis of bladder cancer from urine samples. To bring this technique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation. However, the resolving power of SSCP, a prerequisite basis for reliability required in diagnostics, remains as a challenge for CE systems. We thus focused on this topic and conducted studies on CE instruments equipped with a single capillary or an array of multiple capillaries, using the resolution (Rs) as a quantitative scale for the resolving power. Polymer concentration and buffer are shown to be the decisive parameters. High Rs values of >2.5 are achieved for representative SNPs markers under the optimized conditions, without sacrificing such intrinsic advantages of CE over SGE as the 10-fold quicker migration time and operation that is reproducible, continuous, and automatic. The strategies presented broaden the limits of CE in both the current and related applications.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.     

Fluorescence labeling of human rhinovirus capsid and analysis by capillary electrophoresis

The capsid of human rhinovirus serotype 2, consisting of four viral proteins, was fluorescence-labeled with fluorescein isothiocyanate and analyzed by capillary electrophoresis using UV and laser-induced fluorescence detection. Heat denaturation, proteolytic digestion, and receptor binding were applied for confirmation of the identity of the peak with the labeled virus. Incomplete derivatization with the fluorophore preserved the affinity of the virus for its receptor, indicating that its cell entry pathway is unperturbed by this chemical modification; indeed, an infectivity assay confirms that the labeled virus samples are infectious. The results show that fluorescence labeling of the viral capsid might lead to a valuable probe for studying infection processes in the living cell.     

Determination of electrophoretic mobility distributions through the analysis of individual mitochondrial events by capillary electrophoresis with laser-induced fluorescence detection.

Here we report on the analysis of mitochondrial preparations by capillary electrophoresis with postcolumn laser-induced fluorescence detection. Individual mitochondria are detected by fluorescent labeling with the mitochondrion-selective probe, 10-nonyl acridine orange. Interactions between the organelles and the capillary walls are controlled by derivatization of the capillaries with poly(acryloylaminopropanol). As expected from the presence of charged groups in their outer membranes, isolated mitochondria have intrinsic electrophoretic mobilities. This property may be influenced by variations in size, morphology, membrane composition, and damage caused during the isolation procedure. The mobility distributions of mitochondria isolated from NS1 and CHO cells ranged from -1.2 x 10(-4) to -4.3 x 10(-4) cm2 V(-1) s(-1) and -0.8 x 10(-4) to -4.2 x 10(-4) cm2 V(-1) s(-1), respectively. Furthermore, there seems to be no correlation between the density of the mitochondrial fraction and the resultant electrophoretic mobility distribution. These results suggest a new method for characterization of organelle fractions and for counting individual organelles.     

Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis

The relationship between the electrophoretic mobility, microobs, Stokes radius, rs, ionization state, and solution conformation of the all L-alpha-polypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and several truncated analogues, 3-5, has been investigated under low pH buffer conditions by high-performance capillary zonal electrophoresis (HPCZE) with coated capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentical electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso isomer pair, however, converges. These results indicate that variations in the dipole characteristics of the polypeptide main chain and subtle differences introduced by the spatial constraints of the L-alpha-Pro-->D-alpha-Pro residue replacement lead to differences in the Stokes radii and electrophoretic mobilities of these polypeptides. Since the observed electrophoretic mobilities, microobs, reflect the mean of the mobilities of each charge species participating according to their Stokes radius or their intrinsic charge and mole fraction abundances, the results confirm that polypeptide retro-inverso isomers with unmodified amino and carboxy termini are resolvable. This outcome was achieved despite their notional topographical and conformational similarities as assessed from high-field proton nuclear magnetic resonance (1H NMR) spectroscopy and circular dichroism (CD) spectroscopy.