Behaviour of substituted aminomethylphenol dyes in capillary isoelectric focusing with electroosmotic zone displacement

The behaviour of six substituted aminomethylphenol dyes, having pI values between 5.3 and 10.4, in capillary isoelectric focusing with electroosmotic zone displacement is described. Using untreated fused-silica capillaries and different neutral capillary conditioners in the catholyte, the low-molecular-mass dyes are shown to focus and elute reproducibly in the order of decreasing pI values. In the absence of proteins, the detection times of the dyes are independent of the sampled amount. Hence these substances permit the characterization of the pH gradient produced in this capillary isoelectric focusing method. With concurrent focusing of dyes and test proteins, a macromolecular impact on detection times (reduction of electroosmosis)‘ is revealed. The effect is shown to be dependent on the type and amount of proteins applied and has been observed with three different capillary conditioners. Nevertheless, mapping of the pH gradient with these dyes and determining the pI values of known proteins is shown to provide values in agreement with those in the literature. Hence the substituted aminomethylphenol dyes can be employed as pI markers in capillary isoelectric focusing with electroosmotic zone displacement. Further, focusing and separation of two of the six dyes by preparative recycling free fluid isoelectric focusing is described, illustrating that the substituted aminomethylphenol dyes are also applicable to other free fluid focusing methods.     

Effect of feed zone width on product purity in preparative-scale,continuous free-flow isoelectric focusing separation of enantiomers

The effects of the increased width of the sample feed stream upon the purity of the collected fractions were examined in the continuous free-flow isoelectric focusing separation of the enantiomers of dansyl-tryptophan. Compared to the reference separation obtained with a narrow feed stream introduced through the central sample feed port of the continuous free-flow isoelectric focusing separation unit, the final pH gradient, the position of the enantiomer band centroids and the values of the cumulative product recoveries and cumulative product purities remained essentially identical as the width of the feed band of the racemic sample dissolved in the carrier ampholyte was increased up to the full width of the separation chamber suggesting that the current, limiting practice of narrow, central feed bands can be safely abandoned and dilute feedstock solutions can be utilized in preparative-scale isoelectric focusing enantiomer separations.     

固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

通过聚合物原位聚合反应，制备了部分填充的毛细管整体柱。pH 3~10的载体两性电解质被固化在该毛细管整体柱上。在引入八通进样阀、三通阀和四通连接单元的基础上，构建了适用于固化pH梯度毛细管等电聚焦整体柱（M-IPG）的平台。在蛋白质药物测定过程中，用M-IPG柱和羟丙基纤维素（HPC）涂层毛细管柱同时对曲托珠单抗和依那西谱的等电点进行了测定。结果表明，两种等电聚焦柱都能够同时分离混合蛋白质样品并测定蛋白质类药物中单抗和融合蛋白质的等电点（pI），M-IPG柱所测的pI值与HPC涂层毛细管柱测定的结果基本一致，表明了该柱在进一步构建多维分离平台进行蛋白质组学研究方面的潜力。     

Rapid high-performance isoelectric focusing of monoclonal antibodies in uncoated fused-silica capillaries

Rapid (<5 min) high-performance isoelectric focusing was performed in uncoated fused-silica capillaries to resolve isoforms of monoclonal antibodies and to determine their isoelectric points (pI). The methodology involved the use of a 32 cm (effective length 9 cm)×50 μm I.D. uncoated capillary. (Hydroxypropyl)methyl cellulose was used as an additive to suppress analyte–wall interaction and to precisely control electroosmotic flow so that focusing and mobilization of focused zones past detector occur simultaneously. Urea was included in the separation medium to solubilize the antibodies that precipitated at their point of focusing. The methods with and without urea used ampholytes pH 5–8 to generate a demonstrable linear gradient between pH 5.4 and pH 7.2, based on the separation of various protein standards. Reproducibility [<2% (R.S.D.)] of the migration times (corresponding to the detectable isoforms of the antibodies) was obtained by using two sets of reagents and capillaries on three consecutive days. pI values determined from day-to-day with a reference standard were shown to vary by only 0.01 pH unit. The described capillary isoelectric focusing methods provided a rapid, simple and reproducible way of monitoring micro-heterogeneity and pI of the murine monoclonal antibodies investigated.     

Determination of isoelectric points of acidic and basic proteins by capillary electrophoresis

The isoelectric point (pI) of a protein is of practical importance in many separation procedures, both analytical and preparative. The pI is defined as the pH where the net charge of the protein is zero. Therefore, by plotting the mobilities of the proteins against pH, the intercept at zero mobility should yield the pI value. Isoelectric points have traditionally been determined by isoelectric focusing. In this paper, the potential of capillary electrophoresis as an alternative technique for the determination of pI values of both acidic and basic proteins was investigated. The problem commonly encountered with adsorption of the positively charged proteins with the unprotonated silanol groups of the fused-silica wall is solved by applying a dynamic coating of a polycationic reagent to the wall. The advantages of this technique of determining the pI values are simplicity, speed and minimal sample requirement.     

Improved preparative-scale. continuous, free-flow electrophoretic separation of the enantiomers of terbutaline utilizing equal-but-opposite enantiomer mobilities.

The factors that influence yield and product purity in the continuous, preparative-scale electrophoretic separation of the enantiomers of terbutaline when using the principle of equal-but-opposite effective mobilities were studied. The sodium salt of heptakis-6-sulfato-beta-cyclodextrin was used as the resolving agent, in acidic, isopropanol-containing background electrolytes, in the continuous, free-flow, preparative electrophoretic instrument, the Octopus. By matching the linear velocity of the feed solution to that of the background electrolyte, lateral hydrodynamic dispersion was minimized resulting in a nonelectrophoresed feed band that was only three fractions (about 3 mm) wide as it exited the 0.5 m long separation channel. The multiple of residence time and applied potential was also optimized, constrained by migration of the front of heptakis-6-sulfato-beta-cyclodextrin out of the separation zone, leading to the recovery of 95% of both enantiomers in better than 99.99% purity, at a production rate of 0.1 mg/h.     

Analytical aspects of carrier ampholyte-free isoelectric focusing

The applicability of carrier ampholyte-free isoelectric focusing (CAF-IEF) for analyses of ampholytes is demonstrated. The suggested method is based on the principle of both side regulated ionic matrix in CAF-IEF. A sharp step of pH is created in the column filled with a sample dissolved in a background electrolyte by influence of current and solvolytic fluxes. Here, ampholytes are focused upon. The magnitude of the step, its velocity and direction of its movement can be regulated electrically. In this manner, favorable separation properties of the system can be set up, even during the run. This brings several advantages over conventional methods. The principles of the separation can be easily changed, permitting selective pre-concentration (trapping) of minor components by processing large amounts of a sample to be preformed, effective isotachophoresis or IEF pre-separation and final electrophoretic analysis in one run. Advantages of these combinations are discussed together with the right choice of the working electrolyte. A 1000-fold increase in amount of substance in a column can be achieved for both isotachophoresis and capillary zone electrophoresis combined with CAF-IEF pre-concentration at reasonable working conditions. It enables a limit of detection at the nmol/l level with a concentration factor of about 10(7) to be reached.     

Preparative-scale isoelectric trapping enantiomer separations

The new Gradiflow BF200 IET unit, developed for isoelectric trapping protein separations has been modified and used to carry out preparative-scale enantiomer separations. Hydroxypropyl beta-cyclodextrin was used as the chiral resolving agent to induce an isoelectric point difference between the enantiomers. Three isoelectric membranes with isoelectric points below, in between and above the isoelectric points of the complexed enantiomers were used to trap the separated enantiomers in the anodic and cathodic separation compartments of the Gradiflow BF200 IET apparatus, respectively. The production rates were about 15 times higher than those previously obtained with another isoelectric trapping device and about 30% higher than those obtained in a continuous free-flow electrophoretic device operated in the isoelectric focusing mode. The remarkable separation speed observed in the modified Gradiflow BF200 IET unit is attributed to the favorable interplay of the short electrophoretic transfer distance, the high electric field strength and the large effective surface areas of the isoelectric membranes.     

Preparative-scale isoelectric focusing separation of enantiomers using a multicompartment electrolyzer with isoelectric membranes

The IsoPrime multicompartment electrolyzer, equipped with a series of isoelectric membranes with closely spaced pI values, was used for the first time for the preparative-scale separation of the enantiomers of dansyl phenylalanine with hydroxypropyl beta-cyclodextrin as resolving agent. The final separation conditions could be established easily in three successive experiments by rationally narrowing the pH steps between the neighboring isoelectric membranes. The final separation yielded products with an enantiomeric excess greater than 99.9%, at production rates of about 0.1 mg/h. The greatest experimental difficulty was caused by the relatively high salt content of the hydroxypropyl beta-cyclodextrin used, which resulted in high conductivity and limited the maximum field strength one could use.     

Free-flow zone electrophoresis and isoelectric focusing using a microfabricated glass device with ion permeable membranes

This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow isoelectric focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow isoelectric focusing could be established. Furthermore, the application of hydrodynamic focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow isoelectric focusing employing a transversal voltage of up to 150 V across the separation chamber.     

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.     

Development of a simple ampholyte-free isoelectric focusing slab electrophoresis for protein fractionation

Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. beta-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H(+) and OH(-) ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.     

Use of single-isomer, multiply charge chiral resolving agents for the continuous, preparative-scale electrophoretic separation of enantiomers based on the principle of equal-but-opposite analyte mobilities

A novel approach to continuous, preparative-scale electrophoretic enantiomer separations has been developed based on the observation that stable, equal-but-opposite effective mobilities can be created for the enantiomers of a single-charged analyte by complexing them with a single-isomer, multiply charged resolving agent, provided that the charge of the resolving agent is opposite in sign to that of the uncomplexed analyte enantiomers. When such an analyte-resolving agent system is fed into a continuous, free-flow electrophoretic apparatus, stable, steady-state operating conditions can be established which permit the continuous feeding of the racemic analyte and the collection of pure enantiomers at the opposite sides of the feed stream. This concept is demonstrated via the separation of the enantiomers of terbutaline using heptakis-6-sulfato beta-cyclodextrin as resolving agent, affording production rates as high as 2.8 mg/h in the general-purpose, continuous free-flow electrophoretic system, the Octopus.     

Recent applications of capillary isoelectric focusing

After the advent of capillary isoelectric focusing (CIEF) in the 80's several approaches have been developed in order to use the technique in routine analyses. The recent years showed an extensive increase in the applications of this technique employing its exceptionally high-resolution power. Methodological improvements, as well as hyphenation with other electrophoretic and chromatographic separation procedures, proved the versatility of CIEF in studies of clinically important proteins, recombinant product, cell lysates and other complex mixtures. The combination of CIEF with mass spectrometry detection is one of the major challenges for studying proteomics. This review collected the recent applications of CIEF including innovations in the experimental setup, remedies for the presence of salts in samples, calibration of the pH gradient, carrier ampholyte-free isoelectric focusing, the progress in micropreparation, two-dimensional separations, etc.     

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.     

Multivariate optimisation of a cyclodextrin-assisted-capillary zone electrophoretic method for the separation of torasemide and its metabolites

In this work, a rapid cyclodextrin-assisted capillary electrophoretic method is developed for the separation of the diuretic torasemide and three of its metabolites. Both fractional factorial and central composite designs were employed to optimise the separation method. The factors studied were pH, concentration of methyl-β-cyclodextrin, concentration of the background electrolyte and percentage of acetonitrile as organic modifier. Monitored response was a composite quality response (Q*) which balanced conflicting normalized responses, such as resolution and migration time. Optimal separation of the four studied compounds was achieved in less than 6.5 min, using an electrolyte of 60 mM borate buffer with no organic modifier and 25 mM methyl-β-cyclodextrin concentration adjusted to pH 8.0 at a potential of 30 kV. Detection wavelength and temperature were 197 nm and 20 °C respectively. This work means a significant improvement with regard to a previous separation method for these compounds developed in our laboratory.     

Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing

We describe the purification of a single-strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3TM) designed by Milan Bier et al. for preparative-scale isoelectric focusing. Protein separation takes place entirely in solution, with shear-stabilized laminar flow counteracting convective disturbances generated by the electric field. Conditions for isoelectric focusing were determined by focusing fractions with nuclease activity, following chromatography on DEAE-Sepharose, in analytical gels containing carrier ampholytes. The separation was then scaled up to process larger quantities of protein in the RF3. When partially-purified protein (250 mg, 6700 U/mg) was focused in pH 3-4 carrier ampholytes. 67% of the activity was recovered in pooled peak fractions with a specific activity of 54,000 U/mg protein. Overall, 82% of the activity loaded on the RF3 was recovered. Eliminating two steps prior to isoelectric focusing, and increasing the protein load from 250 mg to 1.2 g, produced an enzyme with a nearly four-fold increase in specific activity (from 4000 U/mg protein to 15,000 U/mg protein) but with unacceptable color. Our results indicate that a high quality enzyme can be prepared in quantity when heat denaturation and ammonium sulfate precipitation are included prior to isoelectric focusing.     

Capillary isoelectric focusing with UV-induced fluorescence detection

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.     

Charge heterogeneity of recombinant pro-urokinase and urinary urokinase, as revealed by isoelectric focusing in immobilized pH gradients

When analysing homogeneous preparations of recombinant pro-urokinase and urinary urokinase by isoelectric focusing (IEF) in immobilized pH gradients, an extreme charge heterogeneity was detected (at least ten major and ten minor bands in the pH range 7–10). This extensive polydispersity was not caused by different degrees of glycosylation, or by IEF artefacts, such as binding to carrier ampholytes or carbamylation by urea. A great part of this heterogeneity could be traced back to the existence of a multitude of protein molecules containing Cys residues at different oxidation levels (-SH, -S-S-, even cysteic acid). Owing to the very large number of Cys residues in pro-urokinase (24 out of a total of 411 amino acids) and to the relatively high pI of its native forms (pI 9.5–9.8; the native form is believed to contain all Cys residues as -S-S- bridges), the presence of SH or cysteic acid residues would increase the negative surface charge, as even SH groups would be extensively ionized. In pro-urokinase, part of the heterogeneity was also due to spontaneous degradation to urokinase and possibly also to cleavage into lower-molecular-mass fragments. When all these causes of heterogeneity were removed, the pI spectrum was reduced to only four, about equally intense, bands. The cause of this residual heterogeneity is unknown.     

Preparative-scale, recirculating, pH-biased binary isoelectric trapping separations

In order to improve the production rates and lower the specific electrophoretic energy consumption values in preparative-scale, recirculating, binary isoelectric trapping separations, we propose to add an auxiliary isoelectric agent to the solution in the anodic separation compartment and another to the solution in the cathodic separation compartment to implement pH-biased isoelectric trapping. The auxiliary isoelectric agents are selected such that they are trapped in the respective anodic and cathodic separation compartments and also, have isoelectric point (pI) values that are different from the pI values of the analytes of interest. By proper selection of the auxiliary isoelectric agents and their concentrations, the analytes of interest can be kept in nonisoelectric, charged state during the entire course of the preparative-scale, recirculating, binary isoelectric trapping separation. This results in higher electrophoretic mobilities and solubilities for the analytes than in their isoelectric or near-isoelectric states, and leads to faster binary isoelectric trapping separations.