Sampling strategies for capillary isoelectric focusing with electroosmotic zone mobilization assessed by high-resolution dynamic computer simulation

The impact of initial sample distribution on separation and focusing of analytes in a pH 3–11 gradient formed by 101 biprotic carrier ampholytes under concomitant electroosmotic displacement was studied by dynamic high-resolution computer simulation. Data obtained with application of the analytes mixed with the carrier ampholytes (as is customarily done), as a short zone within the initial carrier ampholyte zone, sandwiched between zones of carrier ampholytes, or introduced before or after the initial carrier ampholyte zone were compared. With sampling as a short zone within or adjacent to the carrier ampholytes, separation and focusing of analytes is shown to proceed as a cationic, anionic, or mixed process and separation of the analytes is predicted to be much faster than the separation of the carrier components. Thus, after the initial separation, analytes continue to separate and eventually reach their focusing locations. This is different to the double-peak approach to equilibrium that takes place when analytes and carrier ampholytes are applied as a homogenous mixture. Simulation data reveal that sample application between two zones of carrier ampholytes results in the formation of a pH gradient disturbance as the concentration of the carrier ampholytes within the fluid element initially occupied by the sample will be lower compared to the other parts of the gradient. As a consequence thereof, the properties of this region are sample matrix dependent, the pH gradient is flatter, and the region is likely to represent a conductance gap (hot spot). Simulation data suggest that sample placed at the anodic side or at the anodic end of the initial carrier ampholyte zone are the favorable configurations for capillary isoelectric focusing with electroosmotic zone mobilization.     

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.     

The transitional isoelectric focusing process

The transitional isoelectric focusing (IEF) process (the course of pH gradient formation by carrier ampholytes (CAs) and the correlation of the focusing time with CA concentration) were investigated using a whole-column detection capillary isoelectric focusing (CIEF) system. The transitional double-peak phenomenon in IEF was explained as a result of migration of protons from the anodic end and hydroxyl ions from the cathodic end into the separation channel and the higher electric field at both acidic and basic sides of the separation channel. It was observed that focusing times increase logarithmically with CA concentration under a constant applied voltage. The correlation of focusing time with CA concentration was explained by the dependence of the charge-transfer rate on the amount of charged CAs within the separation channel during focusing.     

Divergent-flow isoelectric focusing for separation and preparative analysis of peptides

A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.     

Effects of separation length and voltage on isoelectric focusing in a plastic microfluidic device

This paper describes the investigation on the effects of separation length and voltage on IEF in a plastic microfluidic device. A LIF, whole-channel imaging detection (WCID) system was developed to monitor proteins while they were moving under an electric field. IEF was carried out in a separation medium consisting of carrier ampholytes and a mixture of linear polymers (hydroxyethylcellulose and hydroxypropylcellulose). We found that the IEF separation resolution is essentially independent of separation length when the same voltage is applied, which agrees with the theory. This result supports the notion that IEF in a microfabricated device leads to more rapid analysis without sacrificing the resolving power. A higher separation voltage also brought about more rapid analysis and superior separation resolution. IEF of two proteins (green fluorescence protein and R-phycoerythrin) was achieved in 1.5 min when 500 V was applied across a 1.9-cm channel. We found that a linear relationship exists between the focusing time and the inverse of the electrical field strength. In addition, we confirmed the phenomenon in which the pH gradient was compressed to the middle of a channel, and we found that the relative amount of the gradient compression decreased with the channel length.     

Effects of ampholyte concentration on protein behavior in on-chip isoelectric focusing

The effects of mobility corrections on carrier ampholytes are studied at various ampholyte concentrations to understand protein behavior during IEF. IEF simulations are conducted in the presence of 25 biprotic carrier ampholytes within a pH range of 6-9 after applying the Onsager-Debye-Hückel correction to the carrier ampholytes. Two model proteins with ten charge states but without ionic strength corrections are allowed to focus under an electric field of 300 V/cm in a 1 cm long channel. The IEF simulation results show that higher ionic strengths (50 - 100 mM) cause significant changes in the transient movement as well as the final focused profiles of both ampholytes and proteins. The time required for a single, well-defined peak to form increases with ionic strength when Onsager corrections are applied to the carrier ampholytes. For a particular ampholyte concentration, the space-averaged conductivity does not change during the final focusing stage, but the magnitude of space averaged conductivity is different for different ampholyte concentration. The simulation results also reveal that at steady-state ionic strength profiles remain flat throughout the channel except at the locations of proteins where a significant change in ampholyte concentration is obtained.     

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low‐level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.     

Capillary isoelectric focusing-mass spectrometry for shotgun approach in proteomics

We report on capillary isoelectric focusing-mass spectrometry (CIEF-MS) of complex peptide mixtures in the absence of carrier ampholytes. Furthermore, the use of low concentrations of carrier ampholytes as mere spacers is investigated. Carrier ampholytes are complex mixtures of amphoteric compounds with high buffering capacity. Since all peptides are amphoteric compounds by themselves, the use of carrier ampholytes may be superfluous to establish a stable pH gradient in CIEF analysis of protein digests. Our research showed that when carrier ampholytes are omitted, the analyte ions are not focused at their isoelectric point. The analytes are charged, leading to electrophoretic mobility uncharacteristic for CIEF. The method was tested for a five-protein-mixture at 0.02 mg/mL per protein and 0.05 mg/mL per protein. At the lower concentration, the analytes were stacked during the focusing process in only a limited length of the capillary. Therefore, the higher concentration led to better separation efficiency. It was found that at low concentration (0.20%) the carrier ampholytes could work as spacers. Though it led to sensitivity losses of 15-45%, this was compensated by the higher separation efficiencies seen. The method was evaluated with an eight-protein-mixture, of which all could be identified after performing MS/MS.     

Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples

Efficient separation method is a crucial part of the process in which components of highly complex biological sample are identified and characterized. Based on the principles of recently newly established electrophoretic method called divergent flow IEF (DF IEF), we have tested the DF IEF instrument which is able to operate without the use of background carrier ampholytes. We have verified that during separation and focusing of sample consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be created by autofocusing of the sample components themselves without any addition of carrier ampholytes. In DF IEF, the proteins are separated, desalted and concentrated in one step. The fractions of yeast lysate sample, collected at the DF IEF output and subjected to gel IEF, contained the zones of proteins gradually covering the pI values from 3.7 to 8.5. In our experimental arrangement, the highest number of proteins has been found in fractions with pI values around 5.3 as detected by polyacrylamide gel IEF with CBB staining. During DF IEF, the selected protein bands have been concentrated up to 16.8‐fold.     

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.     

Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients

In this study, metabolically radiolabeled Escherichia coli cell extracts were used to systematically evaluate protein recoveries at each step of two-dimensional (2-D) electrophoresis and using different sample application methods. Sample application using sample cups resulted in better protein recovery compared with sample loading by rehydration when the Multiphor system was used. At least 50% or more of an E. coli extract was lost when high protein amounts (500 microg) were loaded by rehydration using this system, which employs separate holders for rehydration and isoelectric focusing (IEF). In contrast, when the IPGphor system was used, rehydration sample loading consistently yielded the highest overall protein recoveries. These improved protein recoveries were due to integration of rehydration and electrophoretic separation in a single unit. Even at high protein loads (500 microg), less than 15-20% of the proteins were lost when proteins were loaded by rehydration using sample buffer containing 2% carrier ampholytes in the ceramic immobilized pH gradient (IPG) strip holders used for both rehydration and IEF. Regardless of the loading conditions used, carrier ampholytes in the sample buffer increased protein recoveries. Use of thiourea did not significantly affect protein recoveries but did improve protein resolution in 2-D gels as expected. In summary, these results show the best protein recoveries are obtained for all protein loads when samples are applied to IPG strips during rehydration using a single device for both rehydration and IEF. In contrast, the poorest recoveries are obtained when rehydration and IEF are performed in separate devices, and losses increase dramatically with increasing protein loads using this approach.     

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

Instabilities of the pH gradient in carrier ampholyte-based isoelectric focusing: Elucidation of the contributing electrokinetic processes by computer simulation

Electrokinetic processes that lead to pH gradient instabilities in carrier ampholyte-based IEF are reviewed. In addition to electroosmosis, there are four of electrophoretic nature, namely (i) the stabilizing phase with the plateau phenomenon, (ii) the gradual isotachophoretic loss of carrier ampholytes at the two column ends in presence of electrode solutions, (iii) the inequality of the mobilities of positively and negatively charged species of ampholytes, and (iv) the continuous penetration of carbonate from the catholyte into the focusing column. The impact of these factors to cathodic and anodic drifts was analyzed by simulation of carrier ampholyte-based focusing in closed and open columns. Focusing under realistic conditions within a 5 cm long capillary in which three amphoteric low molecular mass dyes were focused in a pH 3–10 gradient formed by 140 carrier ampholytes was investigated. In open columns, electroosmosis displaces the entire gradient toward the cathode or anode whereas the electrophoretic processes act bidirectionally with a transition around pH 4 (drifts for pI > 4 and pI < 4 typically toward the cathode and anode, respectively). The data illustrate that focused zones of carrier ampholytes have an electrophoretic flux and that dynamic simulation can be effectively used to assess the magnitude of each of the electrokinetic destabilizing factors and the resulting drift for a combination of these effects. Predicted drifts of focused marker dyes are compared to those observed experimentally in a setup with coated capillary and whole column optical imaging.     

Improving the detection limit in capillary isotachophoresis using asymmetric neutralisation reaction boundary

An online method involving transient electrokinetic dosing and ITP with neutralization reaction boundary (NRB) and/or carrier ampholyte-free isoelectric focusing (CAF IEF) was developed for the preconcentration, preseparation, and analytical determination of glyphosate in aqueous samples containing low concentrations of the analyte of interest. Various parameters were investigated in the framework of an optimization study with the aim of achieving the maximum concentration limit of detection (cLOD) decrease in minimum time. The proposed method used CAF IEF and/or ITP with NRB. The sample was dosed to the column on the stationary reaction boundary (CAF IEF) and/or moving reaction boundary (ITP with NRB), whereat a sharp pH step exists. Here, charge reversal was due to the ampholytes, and/or acid accumulation occurred because of charge loss. Similarly, the accumulated sample was mobilized with TE and analyzed using classical ITP in the second analytical column. Glyphosate (GLY), the analyte of interest, was chosen as a model substance for ITP with NRB and preconcentration as well as focusing preconcentration and CAF IEF using the asymmetric purpose-built NRB. On one side of the asymmetric boundary was the zone of acidic pH; while the opposite side comprised a neutral/basic non-conductive zone of the ampholyte—in this case, GLY. Such an arrangement enables the use of a lower pH on the acidic side, which allows the focusing of strongly acidic ampholytes and the accumulation of weak acids. The electrolyte composition and the dosing time were optimized, and a 14-fold accumulation was achieved in 25 min compared to that by classical ITP and a 180-fold accumulation was achieved through CAF IEF and preconcentration with a glyphosate sample. Both methods are simple and can be conducted using all commercial ITP systems.     

Evaluation of isoelectric focusing runs as specific resistance/volthour plots.

Isoelectric focusing (IEF) runs, e.g. on ultrathin gels, are characterized by an extensive change of gel electric parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient. A particularly important parameter is the specific resistance rho [Ohm*cm] which has been determined in polymerization mixtures (with and without carrier ampholytes) and in 125 x 0.15 mm ultrathin gels, pH 3-10 with 5% T, 3% C, 5% Servalyt carrier ampholytes, pH 3-10. The starting specific resistance rho of ultra-thin IEF gels, calculated from the geometric gel dimensions and electric current values (V, mA), is in agreement with the data determined directly in 30 mL samples of polymerization mixtures by using a conductivity meter. Electric specific conductivity/Volthour (Vh) plots proved to be a valuable tool for the evaluation of gel systems with and without protein samples during IEP runs. These plots are usually S-shaped, indicating that the key part of pH gradient formation takes places in a relatively short time. A "good" ultrahin gel, after a short lag phase, shows a rapid increase in specific resistance due to a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh. IEF is finished in about 3000 Vh. After prolonged gel storage, and especially in partially dried gels, the electrical parameters approach equilibrium only slowly, as indicated by the relatively shallow slope (8.9 Ohm*cm/Vh). Accordingly, separations need more than 4000 Vh.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autofocusing and ESI-MS analysis of protein digests in a miniaturized multicompartment electrolyzer

Free-solution IEF of protein digests was studied in a newly introduced MicroRotofortrade mark multicompartment electrolyzer. The fractionation was performed in a cylindrical separation chamber divided into ten compartments with or without the addition of carrier ampholytes. In the case of autofocusing mode of operation, the tryptic digest itself served as the mixture of ampholytes leading to the separation of the peptides with well-defined pI's. The focusing process was monitored visually using colored pI markers. The resulting fractions from both modes of the separation were analyzed by CE and electrospray-TOF mass spectrometer using electrospray tips microfabricated in polyimide. Additional experiments, aiming at visualization of the mass flux within the focusing compartments were performed using isotachophoretic migration of color cationic tracers. The study considered the autofocusing of both the peptides with well-defined narrow pI's as well as those showing negligible net charge in a broader pH range. Although not all peptides in the protein digests have well-defined pI's the autofocusing process can preseparate many of them leading to higher S/N in the ESI-MS signals and improved protein sequence coverage.     

Sequential injection setup for capillary isoelectric focusing combined with MS detection

Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection.     

Electrophoretic phenotyping of erythrocyte enzymes.

Erythrocyte acid phosphatase (EAP), esterase D (ESD) and phosphoglucomutase (PGM) phenotypes among the erythrocyte enzyme types of blood groups are surveyed and a modified cellulose acetate membrane isoelectric focusing (CAM-IEF) method for their exploration is described. The phenotyping procedures are usually classified as either equilibrium or non-equilibrium IEF. Equilibrium IEF, which is based on differences in pI values, includes three methods: (i) a narrow pH range of carrier ampholytes, (ii) a relatively narrow pH range of carrier ampholytes containing chemical separators and (iii) immobilized pH gradient gels. Among the three methods, immobilized pH gradients provides a better resolution of isozymes. Conversely, the disadvantages of immobilized pH gradients include longer focusing times and complex gel preparations. Moreover, immobilized pH gradients are unsuitable for stain analysis because of the insensitivity of PGM1 detection. A hybrid IEF system and a commercial immobilized pH gradient dry plate have overcome these problems. However, EAP typing is extremely expensive and ESD typing is not well distinguished by hybrid IEF. As each method has both merits and demerits, the most suitable technique should be selected based on the kind of erythrocyte enzyme types and sample conditions. On the other hand, non-equilibrium IEF is a rapid method because isozymes are detected on the basis of their charge differences under non-equilibrium conditions. Moreover, the appropriate addition separators increases the charge difference and provides a good resolution within a shorter time. Addition of more separators produces a narrow pH range in the gel and takes a substantially longer time to reach the optimum pH range for charge difference.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Modeling and simulation of IEF in 2-D microgeometries

A 2-D finite-volume model is developed to simulate nonlinear IEF in complex microgeometries. This mathematical model is formulated based on the mass conservation and ionic dissociation relations of amphoteric macromolecules, charge conservation, and the electroneutrality condition. Based on the 2-D model, three different separation cases are studied: an IPG in a planar channel, an ampholyte-based pH gradient in a planar channel, and an ampholyte-based pH gradient in a contraction-expansion channel. In the IPG case, cacodylic acid (pK(1) = 6.21) and Tris (pK(1) = 8.3) are used as the acid and base, respectively, to validate the 2-D IEF model. In the ampholyte-based pH gradient cases, IEF is performed in the pH range, 6.21-8.3 using 10 ampholytes in the planar channel and 20 ampholytes in the contraction-expansion channel. The numerical results reveal different focusing efficiencies and resolution in the narrow and wide sections of the contraction-expansion channel. To explain this, the expressions for separation resolution and peak concentrations of separands in the contraction-expansion channel are presented in terms of the channel shape factor. In a 2-D planar channel, a focused band remains straight all the time. However, in a contraction-expansion channel, initially straight bands take on a crescent profile as they pass through the trapezoidal sections joining the contraction and expansion sections.