Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis

Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.     

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Antibody fragment separations by capillary zone electrophoresis

This study investigated methods of improving the separation and identification of an IgA antibody, McPC603, and its pepsin fragments. The problem presented by purification of antibody fragments (Fabs) and the antibody light chain required accurate and informative analysis of highly hydrophobic proteins, which can polymerize and fold to form secondary structures. Capillary zone electrophoresis (CZE) permits the separation of peptides and small proteins by a method which is orthogonal to the traditional method of reversed-phase HPLC. To facilitate planned studies of the antibody's biological activity, our buffer composition was kept as simple as possible. During CZE analysis, if the buffer pH is below the isoelectric point of the protein, or the protein is large (with a heterogeneous distribution of surface charges), it can irreversibly bind to the capillary wall unless the capillary is coated. We found that C₁-coatings in RP-capillaries at pH 9.5 adequately prevented the antibody fragments from binding to the wall. However, the coating did not remain stable at such high pH, so different conditions were sought. We achieved adequate separations in several buffers at nearly physiological pH, in a bare silica capillary which had been coated once with a soluble cationic polymer coating (Micro-Coat applied during column conditioning). Antibody electropherograms changed depending on the type of inorganic buffer salt used in a separation. Phosphate binds to the antigen-binding site of the IgA with low affinity, and interesting effects were observed in separations using phosphate buffer. These effects will be discussed.     

Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels

Due to their heterogeneity and huge differences in abundance, the detection and identification of all proteins expressed in eukaryotic cells and tissues is a major challenge in proteome analysis. Currently the most promising approaches are sample prefractionation procedures prior to narrow pH range two-dimensional gel electrophoresis (IPG-Dalt) to reduce the complexity of the sample and to enrich for low abundance proteins. We recently developed a simple, cheap and rapid sample prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated gels. Complex sample mixtures are prefractionated in Sephadex gels containing urea, zwitterionic detergents, dithiothreitol and carrier ampholytes. After IEF, up to ten gel fractions alongside the pH gradient are removed with a spatula and directly applied onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of two-dimensional (2-D) gel electrophoresis. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and the full compatibility with subsequent IPG-IEF, since the prefactionated samples are not eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with subsequent IPG-IEF. Prefractionation allows loading of higher protein amounts within the separation range applied to 2-D gels and facilitates the detection of less abundant proteins. Also, this system is highly flexibile, since it allows small scale and large scale runs, and separation of different samples at the same time. In the current study, this technology has been successfully applied for prefractionation of mouse liver proteins prior to narrow pH range IPG-Dalt.     

Use of polybuffer as carrier ampholytes in mixed-bed Immobiline gels for isoelectric focusing

In mixed-bed, carrier ampholyte-Immobiline gels, a primary, insolubilized pH gradient is admixed with a secondary, soluble pH gradient generated by amphoteric buffers. The latter are the standard carrier ampholytes (e.g. Ampholine, Pharmalyte, Biolyte, Servalyte), used in conventional isoelectric focusing, admixed to Immobiline gels in levels of approximately 0.5-1%. It is here shown that polybuffers 96 (covering the pH 6-9 range) and 74 (covering the pH 4-7 interval) used as eluents in chromatofocusing, can effectively substitute the standard carrier ampholytes with considerable savings (they are 1/16th as expensive as the latter chemicals).     

Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis

A series of 10 synthetic peptides containing varying degrees of charge and hydrophobicity was used to study the effects of peptide composition and buffer pH on the selectivity of separations by capillary zone electrophoresis (CZE). A simple model is used to explain the effect of buffer pH on the separation. It was found that pH is an important parameter affecting the selectivity of CZE separations. Furthermore, it is shown that the selectivity of the separation is such that peptides differing in neutral amino acid composition can be resolved, and that even differences in a peptide's amino acid sequence can be detected. A protease digest of beta-lactoglobulin A is shown as a practical example of a separation of a complex peptide mixture.     

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).     

Two-dimensional maps in the most extended (pH 2.5-11) immobilized pH gradient interval

In conventional isoelectric focusing in soluble, amphoteric buffers, it has been quite difficult to produce two-dimensional (2-D) separations in pH intervals greater than pH 4-8. In general more alkaline proteins were analyzed by non-equilibrium IEF in the first dimension. Even with the advent of immobilized pH gradients (IPG), separations could be extended to pH gradients not wider than pH 3-10, due to a lack of suitable buffers. Since more acidic and more alkaline acrylamido buffers have recently been synthesized, we have been able to optimize what is believed to be the widest possible immobilized pH gradient, a pH 2.5-11 span. We report here for the first time 2-D separations of total tissue lysates in such extended pH 2.5-11 gradients. It appears that, with the IPG technique, close to 100% of all possible cell products can be displayed in a single 2-D map.     

Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.     

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow range ampholytes prepared in this way are comparable to commercial ampholyte preparation as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.     

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.     

Determination of L-ascorbic acid in Lycopersicon fruits by capillary zone electrophoresis

This study shows an improved method for the determination of L-ascorbic acid (l-AA) in fruits of Lycopersicon by capillary zone electrophoresis (CZE). Two backgrounds electrolytes (BGEs) have been tested: (i) 400 mM borate at pH 8.0 and 1 x 10(-2)% hexadimethrine bromide, for the separation of Eulycopersicon subgenus species; and (ii) as in BGE(i) but supplemented with 20% (v/v) acetonitrile, for the separation of species of the Eriopersicon subgenus. The present procedures were compared with two routine methods-enzymatic assay and potentiometric titration with 2,6-dichlorophenol-indophenol. While these routine methods presented some difficulties in quantifying l-AA in several Lycopersicon fruits, CZE was successfully applied in all the analyzed samples. The proposed CZE protocols give lower detection limits (<0.4 microg ml(-1)); are cheaper, quicker, and highly reproducible; and can be applied to analyze large series of samples (ca. 50 samples per day) which is utmost importance, not only in screening trials for internal quality and tomato breeding programs, but also in systematic and routine characterization of Lycopersicon fruits.     

Evaluation of the efficiency of extraction of ultraviolet-absorbing pollen allergens and organic pollutants from airborne dust samples by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), have been used for evaluation of the efficiency of different extraction agents applied to the extraction of pollen allergens and organic pollutants from dust samples collected during different periods (before, during and after pollen seasons) and in different locations in air-filtration devices (car-traffic tunnel in Prague and a metro station in Paris). Water and acetic acid extracts were analyzed by CZE using acetic acid as background electrolyte (BGE). Water and alkaline water-SDS-buffer extracts were analyzed by MEKC in Tris-phosphate BGE with anionic detergent sodium dodecylsulfate (SDS) micellar pseudophase. More material was extracted and more components were found in the water-buffer extracts than in the water extracts, and better resolution of the components was achieved by MEKC than by CZE. Significant differences have been found in the analyses of dust extracts of different origin. More material and more components have been found in the extracts of the dust collected in the pollen-rich period (March, April) than in the pollen-free period (December, January).     

Analysis of liquid extracts from tree and grass pollens by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE), micellar electrokinetic chromatography (CMEKC) and isotachophoresis (CITP), have been used for analysis of water and water-buffer extracts from tree-common birch (Betula verrucosa) and grass-orchardgrass (Dactylis glomerata) pollen samples. Water extracts were analyzed by CZE using acetic acid as background electrolyte (BGE), by CMEKC in tris-phosphate BGE with anionic detergent sodium dodecyl sulfate (SDS) micellar pseudophase (TP-SDS) and by CITP in cationic mode with leading/terminating cations K+/BALA+ (beta-alanine (BALA)) and in anionic mode with leading/terminating anions Cl-/MES- (2-(N-morpholino)ethanesulphonic acid (MES)). Moreover, acetic acid extracts were analyzed by CZE using acetic acid as BGE, and alkaline water-SDS-buffer extracts were analyzed by CMEKC using TP-SDS as BGE. Extracted amounts of pollen allergens and other UV-absorbing compounds and the number of resolved components were evaluated from CZE, CMEKC and CITP analyses of the liquid extracts. Larger amounts of UV-absorbing material were found in the water-buffer pollen extracts than in the water extracts. More UV-absorbing material was found in all extracts from D. glomerata pollen than in relevant extracts from B. verrucosa pollen. It was found by CITP that the extracted amounts of anionic components and their number were much higher than those of cationic components. Concentrations of some inorganic ions (e.g. Cl-, K+, Na+, Ca2+) in pollen samples were also determined by CITP.     

Separation of nucleosides and nucleotides by reversed-phase high-performance liquid chromatography with volatile buffers allowing sample recovery

Reversed-phase high-performance liquid chromatography using a C18 column with volatile buffers as the eluant was applied to the separation of a number of nucleosides and nucleotides. Groups of seven nucleosides and five nucleoside monophosphates were separated isocratically employing 0.1 M trimethylammonium acetate and 2% acetonitrile at pH 7.0. Groups of seven nucleoside diphosphates and seven nucleoside triphosphates were separated with 0.1 M triethylammonium bicarbonate and 2% acetonitrile titrated to a pH of 7.1 with acetic acid. The techniques described give resolution and separations comparable to nonvolatile buffers. Moreover, the eluant trimethylammonium acetate or triethylammonium bicarbonate buffer can easily be removed in vacuo from the column effluent, making the technique useful for preparative separations of these compounds. The observed elution pattern of nucleoside phosphates suggests that "paired-ion" chromatography is involved in the separation.     

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization

Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.     

Monitoring equilibria and kinetics of protein folding/unfolding reactions by capillary zone electrophoresis

A method is described here for studying conformational transitions of proteins due to denaturing agents: capillary zone electrophoresis (CZE) in acidic, isoelectric buffers. The sample is run in 50 mM isoelectric glutamic acid (pH = pI = 3.2) added with 1 mM oligoamine (tetraethylene pentamine) for quenching protein interaction to the capillary wall (final pH = 3.3). Muscle acylphosphatase (AcP), in this buffer, exhibited a free solution mobility of 2.63 x 10(-4) cm(2) V(-1) s(-1). By studying the unfolding kinetics, as a function of time of incubation in 7 M urea, it was possible to measure the rate constant of the unfolding reaction, estimated to be 0.00030+/-0.00006 s(-1). The same measurements, when repeated via spectroscopic monitoring of intrinsic fluorescence, gave a value of 0.00034+/-0.00002 s(-1), thus in excellent agreement with CZE data. By equilibrium unfolding CZE studies, it was possible to construct the typical sigmoidal transition of unfolding vs urea molarity: the midpoint of this transition, at which the folded and unfolded states should be equally populated, was estimated to be at 4.56 M urea. Similar experiments by fluorometric analysis gave a value of 4.60 M urea as midpoint of the unfolding curve.     

Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis

One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.     

Scanning Isoelectric Focusing and Isotachophoresis of Clostridium perfringens Type A Enterotoxin

Fractionation of highly purified Cl. perfringens type A enterotoxin by scanning isoelectric focusing (SIF) and isotachophoresis (IT) in polyacrylamide gels is described for the first time. The use of 2% ampholytes pH 3–6 allowed the separation of enterotoxin into 2 species. The major component had an isoelectric point of 4·5 and possessed antigenic as well as functional activity. The minor component of enterotoxin, at equivalent concentrations, was devoid of any demonstrable biological activity had an isoelectric point of 4·6 and appeared to represent approximately 15% of the purified enterotoxin. With ampholytes pH 3·5–10 the minor and major components were focused at different times than when ampholine pH 3–6 was employed. Electrofocusing of enterotoxin in the presence of 6 M-urea did not alter the SIF pattern. During IT the major component of enterotoxin migrated ahead of the minor component. The 2 proteins were completely separated. Isotachophoretic separations required 0·023 M-phosphate pH 6·0 as the leading ion, 0·079 M-Tris as the counter-ion, 0·2 M-glycine (in Tris pH 8·1) as the terminating ion, 30 γ carrier ampholytes pH 3·5–10, 263 μg enterotoxin, 4% acrylamide and a current of 5 mA per gel column.