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)     

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10-100 fold in comparison with conventional IEF.     

CE determination of the thermodynamic pKa values and limiting ionic mobilities of 14 low molecular mass UV absorbing ampholytes for accurate characterization of the pH gradient in carrier ampholytes-based IEF and its numeric simulation

Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpK_a = pKa_monoanion ‒ pKa_monocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download ), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pK_a values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pK_a values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pK_a and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download ).     

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.     

Isoelectric focusing field-flow fractionation IV. Investigations on protein separations in the trapezoidal cross-section channel

The first separation of three proteins (horse spleen ferritin, equine myoglobin and horse heart cytochrome c) by isoelectric focusing field-flow fractionation in a trapezoidal cross-section channel of 0.875 ml volume and 25 cm length is reported. Separation and elution are shown to proceed within about 1 h at a power application of about 1 W. The separation of the three proteins is demonstrated to be dependent on applied electric power, carrier ampholyte concentration and the concentrations of anolyte and catholyte. It follows from these data that the resolution is improved with increasing carrier ampholyte concentration and/or decreasing concentrations of the electrode solutions. The experimentally observed effects are in agreement with predictions made by computer simulation.     

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.     

Isoelectric focusing in immobilized pH gradients with carrier ampholytes added for high-resolution phenotyping of bovine beta-lactoglobulins: characterization of a new genetic variant

The genetic variants of bovine beta-lactoglobulin (beta-lg) from the "Murnau-Werdenfelser" breed were analyzed in three different isoelectric focusing (IEF) systems. While carrier ampholyte IEF with a pH gradient of 0.2 pH/cm did not resolve the new variant W from the B variant and IEF in immobobilized pH gradients (IPG) with 0.1 pH/cm only partially resolved it, adequate separation was achieved with IPG-IEF in a pH 5.25-pH 5.7 gradient, in presence of 0.8 % w/v carrier ampholytes, both over a 10 and 17 cm separation distance. Apparent isoelectric points (pI's) and genetic frequencies (f) were as follows: beta-lg A, pI = 5.370, f = 0.364; beta-lg B, pI = 5.485, f = 0.480; beta-lg W, pI = 5.492, f = 0.076; and beta-lg D, pI = 5.610, f = 0.080. The small difference of delta pI = 0.007 between beta-lg B and beta-lg W respectively, seems to originate from a "silent" substitution of neutral amino acid residues as compared to the larger delta pI's of the other genetic variants of beta-lg, which result from substitution of charged amino acids.     

Ionic binding properties of carrier ampholytes

Radioactive ampholytes were synthesized with specific activity of 638 microCi/g. These were used in studies of ampholyte binding to target proteins under non-ionic conditions. These radioactive ampholytes bound to target proteins but were dissociable in sodium chloride solutions with dissociation occurring in a concentration dependent way. The ampholytes could be dissociated from target molecules using excess unlabelled ampholytes synthesized in the laboratory as well as commercial ampholytes. Radioactive ampholytes were bound to target proteins with different isoelectric points and the bound ampholytes were eluted and analyzed by recycling isoelectric focusing. The results showed that acidic proteins bound basic ampholytes and basic proteins bound acidic ampholytes. Acidic radioactive ampholytes were selectively bound by Sephacryl S-200 and ampholyte exchange from protein to Sephacryl S-200 was shown.     

High-resolution computer simulation of the dynamics of isoelectric focusing: in quest of more realistic input parameters for carrier ampholytes

The impact of the systematic variation of either DeltapK(a) or mobility of 140 biprotic carrier ampholytes on the conductivity profile of a pH 3-10 gradient was studied by dynamic computer simulation. A configuration with the greatest DeltapK(a) in the pH 6-7 range and uniform mobilities produced a conductivity profile consistent with that which is experimentally observed. A similar result was observed when the neutral (pI = 7) ampholyte is assigned the lowest mobility and mobilities of the other carriers are systematically increased as their pI's recede from 7. When equal DeltapK(a) values and mobilities are assigned to all ampholytes a conductivity plateau in the pH 5-9 region is produced which does not reflect what is seen experimentally. The variation in DeltapK(a) values is considered to most accurately reflect the electrochemical parameters of commercially available mixtures of carrier ampholytes. Simulations with unequal mobilities of the cationic and anionic species of the carrier ampholytes show either cathodic (greater mobility of the cationic species) or anodic (greater mobility of the anionic species) drifts of the pH gradient. The simulated cationic drifts compare well to those observed experimentally in a capillary in which the focusing of three dyes was followed by whole column optical imaging. The cathodic drift flattens the acidic portion of the gradient and steepens the basic part. This phenomenon is an additional argument against the notion that focused zones of carrier ampholytes have no electrophoretic flux.     

Distortion of the electrophoretic titration curves of some proteins

The electrophoretic titration curves of complex mixtures of vitamin K-dependent human blood proteins and proteins of Bothrops asper venom were investigated. In both protein mixtures some curves exhibited marked distortions such as additional maxima and minima when Pharmalyte 3-10 carrier ampholytes were used for isoelectric focusing in agarose gels. The distortions result from an unspecific interactions between some carrier ampholyte constituents with particular proteins. The interacting carrier ampholyte components could be completely removed by binding to albumin and ultrafiltration through a UM-2 Amicon membrane with resultant regular titration curves. The interacting carrier ampholyte species were only partially removed by ultrafiltration through a UM-2 membrane without incubation with albumin.     

Loading capacity of carrier ampholytes--based buffers in capillary electrophoresis

Narrow pH cuts of carrier ampholytes (CAs), originally designed for IEF, have been used as BGEs in CE. Their physicochemical properties, rather high buffering capacity and low conductivity, allow very efficient protein separations under high electric field strength. Due to their isoelectric properties, CA BGEs are expected to present a low ionic concentration and consequently a low loading capacity. In this study, we developed a simple method that allows the estimation of the loading capacity of a UV-absorbing BGE by CE. We first characterized in terms of loading capacity, classical ammediol-chromate UV-absorbing BGEs and a 10 mM histidine solution, a classical isoelectric buffer. Then, the loading capacity of four different CA-based BGEs has been assessed. Experimental results have shown that the CA-based buffers were presenting a rather high loading capacity, comparable to classical buffer ones and far higher than the one of the 10 mM histidine solution.     

Carrier ampholyte‐free free‐flow isoelectric focusing for separation of protein

Free‐flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home‐made carrier ampholyte‐free FFIEF system was constructed via orientated migration of H⁺ and OH^? provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R² = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte‐free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI‐TOF‐MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15 mmol/L Tris‐acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.     

Evidence against the occurrence of artifacts due to carrier ampholyte-protein binding during isoelectric focusing.

The formation of irreversible complexes between carrier ampholyte components and proteins was investigated by gel filtration of mixtures of proteins and radioactively labelled ampholytes. Experiments were performed both with purified proteins (albumin, ferritin, beta-glucuronidase) and with a complex mixture of proteins (serum); in no case was binding of ampholytes to proteins detected. Thus the results argue against the occurrence in isoelectric focusing of proteins of artifacts due to such complex formation.     

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.     

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.     

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.     

Simul 5 - free dynamic simulator of electrophoresis

We introduce the mathematical model of electromigration of electrolytes in free solution together with free software Simul, version 5, designed for simulation of electrophoresis. The mathematical model is based on principles of mass conservation, acid-base equilibria, and electroneutrality. It accounts for any number of multivalent electrolytes or ampholytes and yields a complete picture about dynamics of electromigration and diffusion in the separation channel. Additionally, the model accounts for the influence of ionic strength on ionic mobilities and electrolyte activities. The typical use of Simul is: inspection of system peaks (zones), stacking and preconcentrating analytes, resonance phenomena, and optimization of separation conditions, in either CZE, ITP, or IEF.     

Preparative isoelectric focusing of proteins using binary buffers in a vortex-stabilized, free-flow apparatus

Recombinant proteins are often produced as isoforms with different kinds and amounts of post-translational modifications that alter their function. Isoelectric focusing in shallow pH gradients, less than 0.5 pH/cm, might be capable of fractionating these isoforms. The synthetic carrier ampholyte mixtures typically used to generate these pH gradients are expensive and may adversely interact with proteins. Using defined buffers instead of synthetic carrier ampholytes reduces these problems. We tested two defined buffer systems in a vortex-stabilized electrophoresis device to see if they could form shallow pH gradients useful for separating isoforms. These pH gradients were formed by pouring a two-component concentration gradient. The poured gradients were smooth, reproducible, and stable for at least 1.5 h at 5 kV. One poured gradient focused 20 mg of cytochrome c. A second poured gradient separated glucose oxidase from amyloglucosidase. The breadth of the amyloglucosidase band indicates that the shallow, poured pH gradients can only partially separate protein isoforms at 10 kV. Proteins with pI < 0.2 pH units apart will have overlapping bands in these shallow, poured pH gradients.     

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.     

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.