Isoelectric Buffers Research Articles

Studies of Histidine As a Suitable Isoelectric Buffer for Tryptic Digestion and Isoelectric Trapping Fractionation Followed by Capillary Electrophoresis–Mass Spectrometry for Proteomic Analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.

Recent advances in electrophoretic techniques for the characterization of protein biomolecules: A poker of aces

The four classical modes of electrophoresis of protein molecules (sodium dodecyl sulphate electrophoresis, SDS-PAGE, isoelectric focusing, IEF, and immobilized pH gradients, IPGs, two-dimensional maps, 2D, and capillary electrophoresis, CE) are here reviewed, with special emphasis on recent innovations. Thus, in the case of SDS-PAGE, a novel method, consisting in focusing SDS-protein micelles against a gradient of cationic charges grafted onto a polyacrylamide gel is presented. In the case of IEF, the recent decoding of the structure, polydispersity, molecular mass distribution and buffering properties of the soluble carrier ampholyte buffers are here discussed. In regard to two dimensional mapping, recent instrumentation for performing 2D maps in horizontal, large gel slabs (up to 30 cm × 40 cm) and in a radial format for the SDS dimension is here evaluated. Finally, in the case of CE, three major applications are presented: a thorough study of capillary IEF and of all experimental variables, a method of importance in screening of rDNA products; the possibility of running proteins and peptide separations in very acidic, amphoteric, isoelectric buffers in absence of any capillary coating; finally, the possibility of producing a facile, user friendly, covalent coating of the wall silanols via bonding of quaternarized piperazines endowed with an iodinated tail. In acidic, volatile buffers, such protein/peptide runs can be directly interfaced with mass spectrometry instrumentation.

Identification and characterization of high‐molecular‐weight secalins from triticale seeds by capillary zone electrophoresis

A rapid and reliable method for separation and characterization of the variability of high-molecular-weight secalin subunits (HMW-SS) in hexaploid triticale (x Triticosecale Wittmack) by CZE has been developed. In this method, a mixture of two poly(ethylene oxide) polymers differing in molecular weight and a high concentration of ACN in isoelectric buffer was applied as the running electrolyte. For dynamic coating of the capillary inner wall, a low-concentration mixture of poly(vinylpyrrolidone) and hydroxypropylmethylcellulose was employed. Wide allelic variations in rye HMW-SS composition, including some novel x- and y-type HMW-SS, were detected by CZE. The CZE electropherograms of HMW-SS showed two groups of peaks in accordance with y- and x-type subunits, with migration times of 8.0-8.8 and 11.0-13.3 min, respectively. HMW-SS differed in migration times from the simultaneously resolved HMW glutenin subunits, but frequently had very similar electrophoretic mobilities during separation by SDS-PAGE. Each of the two rye subunits 2r and 6.5r detected by SDS-PAGE represents in fact two subunits (5.1r or 5.2r, and 6.4r or 6.5r, respectively). After analyzing 106 European triticale cultivars, 12 HMW-SS were identified (six x-type and six y-type). They form six allelic variants of these subunits. The simultaneous separation and identification of triticale HMW glutenin and secalin subunits by CZE is an efficient alternative to SDS-PAGE and should facilitate breeding of valuable cultivars.

CZE for glycoform profiling and quality assessment of recombinant human interleukin‐7

The aim of the present work was to develop a simple high-resolution CZE method for glycoform profiling and quality assessment of recombinant human interleukin-7 (rhIL-7) produced in Chinese hamster ovary cells. Classical and isoelectric buffers and several monoamines used as alkaline component of BGE at very low concentration have been investigated in order to optimize the resolution. The best separation was achieved using a fused-silica capillary and a buffer composed of 25 mM citrate/triethanolamine at pH 2.6. Under these acidic conditions, triethanolamine was able to reverse EOF favorably and to limit rhIL-7 adsorption. This method allowed the separation of four groups of peaks ranging from low to high-sialylated glycoforms. An extensive study on inter-run rinsing procedures has been conducted. Rinsing with 50 mM SDS was retained to achieve the optimal repeatability. Excellent intermediate precision was obtained for migration time (RSD < 0.6%), while RSD for intraday studies were only less than 2.9%. Satisfactory inter and intraday repeatabilities were also observed for relative peak area. We finally demonstrated that reliable information could be obtained to address comparability studies and demonstrate batch-to-batch consistency of biomanufactured rhIL-7.

Detection of High Molecular Weight Glutenin Subunits in Triticale (× Triticosecale Wittm.) Cultivars by Capillary Zone Electrophoresis

An improved method for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in hexaploid triticale by capillary zone electrophoresis (CZE) was developed. A low-concentrate mixture of hydrophilic polymers, poly(vinylpyrrolidone) (PVP) and hydroxypropylmethylcellulose (HPMC), in an isoelectric buffer was employed for dynamic coating of the capillary inner wall. In separation buffer PVP with lower concentrated poly(ethylene oxide) (PEO) was replaced. The CZE electropherograms of HMW-GS showed two group peaks in accordance with x- and y-type subunits with migration times of 6.8-7.8 and 8.4-11.5 min, respectively. In total, 14 HMW subunits (2 subunits encoded by Glu-A1 locus and 12 by Glu-B1) were identified. The CZE analyses revealed that each of the subunits Bx7 and By8 determined by SDS-PAGE makes up three subunits (Bx6.8, Bx7, and Bx7* and By8, By8*, and new By8**, respectively), with different migration times. It was also shown that the subunits By18 and By20 in triticale determined by SDS-PAGE have different migration times in comparison with the same subunits in bread wheat. For these new HMW-GS, the following names were assigned: By18* instead of By18 and By20* instead of By20. The presented CZE method is an efficient alternative to the SDS-PAGE procedure for early selection of useful triticale genotypes with good breadmaking quality.

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.

Capillary electrophoresis for monitoring dityrosine and 3-bromotyrosine synthesis

Protein oxidation affects the structure of many amino acids. Variants of tyrosine are increasingly important in medical and food sciences. The synthesis of standards is essential for monitoring the disease state of patients with various illnesses and the quality of a number of food products. A method for monitoring standard synthesis of dityrosine and 3-bromotyrsoine from tyrosine using capillary electrophoresis (CE) is presented. Optimum separation was achieved using an isoelectric buffer consisting of 100 mM iminodiacetic acid (IDA) + 75 mM lauryl sulfobetaine (SB 3-12) + 0.02% hydroxypropyl methylcellulose (HPMC) in a 27 cm × 75 μm capillary at 22 kV and 45 °C. Using these conditions the tyrosine adducts could be easily separated in less than 4 min. The resolution of the CE method was similar to HPLC separations, but analysis time was distinctly shorter.

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 buffers, part 3: Determination of pKa and pI values of diamino sulfate carrier ampholytes by indirect UV‐detection capillary electrophoresis

Ampholytes with close pK(a) values (i.e., good carrier ampholytes (CAs)) are needed as buffers in pH-biased isoelectric trapping (IET) separations. The syntheses of two families of such good CAs were reported recently. Members of the family of diamino sulfate ampholytes (first series) had pI values in the 5.7 < pI < 9.0 range. Members of the family of quaternary ammonium dicarboxylic acid ampholytes (second series) had pI values in the pI < 4.3 range. To further characterize the diamino sulfate ampholytes, their effective mobilities were measured by indirect UV-absorbance detection capillary electrophoresis in a series of background electrolytes (BGEs) with different pH values. The pK(a) and limiting ionic mobility values of the CAs were obtained by fitting these mobility values, as a function of the pH and the ionic strength of the BGEs, to the theoretical mobility expression. These diamino sulfates complete the list of CAs suitable for IET separations.

Synthesis and characterization of quaternary ammonium dicarboxylic acid isoelectric buffers and their use in pH-biased isoelectric trapping separations

Two approaches are described in this paper for the synthesis of isoelectric buffers that have pI values in the 1.5 < pI < 4.3 range. The first synthesis relies on the alkylation of existing aminodicarboxylic acids and recovery of the ampholyte as an inner salt. The second synthesis method forms low-pI ampholytes by reacting a secondary amine with two equivalents of an alkylester of a haloalkanecarboxylic acid, followed by hydrolysis of the intermediate in an alkaline solution and recovery of the ampholyte as an inner salt. The new ampholytes have been analytically characterized by capillary electrophoresis, high-resolution electrospray ionization-mass spectrometry, one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. The isoionic solutions of the new ampholytes have high buffering capacity and conductivity, making them good pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping separations.

Analysis and separation of enkephalin and dalargin analogues and fragments by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been applied to qualitative and quantitative analysis and separation of synthetic analogues and fragments of enkephalins ([Leu 5]enkephalin, H-Tyr-Gly-Gly-Phe-Leu-OH, [Met 5]enkephalin, H-Tyr-Gly-Gly-Phe-Met-OH), and dalargin (H-Tyr- d-Ala-Gly-Phe-Leu-Arg-OH), biologically active peptides with morphin-like effects acting as ligands for the opiate receptors in the brain. These oligopeptides (dipeptides to hexapeptides) were analyzed as cations in two acidic background electrolytes (BGEs), BGE I (100 mM H 3PO 4, 50 mM Tris, pH 2.25), BGE II (100 mM iminodiacetic acid, pH 2.30), and both as cations and anions in alkaline BGE IV (40 mM Tris, 40 mM Tricine, pH 8.10). Purity degrees of peptides, expressed in three different ways (relative peak height, relative peak area and relative corrected peak area), were determined by their CZE analyses in the above BGEs, and their values were compared with respect to the peak shapes and migration times of the main synthetic products and their admixtures. Selected analogues and fragments of enkephalins and dalargin were successfully separated by CZE in acidic isoelectric buffers, 100 and 200 mM iminodiacetic acid, pH 2.30 and 2.32, respectively. The effective electrophoretic mobilities at standard temperature 25 °C, and effective and specific charges of all analyzed peptides in the above three BGEs were determined. Correlation between effective electrophoretic mobility of the analyzed peptides and their charge and size (relative molecular mass) was investigated, which revealed different molecular shape of analyzed peptides in acidic and alkaline BGEs. In addition, the selected characteristics of the UV-absorption detector (noise, signal to noise ratio, sensitivity, and limits of detection and quantification) were determined.

A family of high-buffering capacity diamino sulfate isoelectric buffers for pH-biased isoelectric trapping separations

pH-biased isoelectric trapping separations are hindered by the lack of suitable isoelectric buffers with pI values in the 5.8 < pI range. Two generic approaches are described here for the cost-effective synthesis of a family of diamino sulfate buffers that have high buffering capacities in their isoelectric state: the first approach relies on the sulfation of existing, commercially available diamino alcohol intermediates, the second approach calls for the synthesis of diamino alcohols from epichlorohydrin and widely available secondary amines, and subsequent sulfation of the new diamino alcohol. The diamino sulfate buffers are recovered in isoelectric state, in high purity. Four members of the family having pI values in the 5.8 < pI < 8.9 range have been synthesized, analytically characterized by capillary electrophoresis (CE), electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), 1-D and 2-D nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. All four diamino sulfates have been successfully used as pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping protein separations.

Preparative-scale fractionation by isoelectric trapping under nondenaturing conditions: Separation of egg white protein isoforms on a modified Gradiflow unit

pH-biased isoelectric trapping was used to separate proteins from egg white at the preparative level (80 mg), into discrete protein fractions based on isoelectric point. The problems of isoelectric precipitation that are common for the separation of complex protein mixtures under isoelectric conditions were mitigated by using single-component isoelectric buffers within the sample separation compartments. This combined with the mild process conditions of the Gradiflow unit that was modified for binary isoelectric trapping separations, ensured that biological activity was maintained. This was verified by measurement of the trypsin protease inhibitory activity of the extract and separated fractions. Furthermore, the high resolving power of this system under preparative conditions was demonstrated by separation of three protein isoforms using isoelectric membranes with differences of 0.025 pH units from each other.

Isoelectric Buffers for Capillary Electrophoresis. 2. Bismorpholine Derivative of a Carboxylic Acid as a Low Molecular Weight Isoelectric Buffer

A new compound class of synthetic isoelectric buffers is introduced, designed as a small molecule with one fully or prevailingly dissociated acidic group (such as sulfonic or carboxylic) and two partly pronated (buffering) basic amino groups attached onto a hydrophilic UV-transparent backbone. As an example, a new isoelectric compound 2,2-bis(4-morpholinylmethyl)propanoic acid (BMMPA) was synthesized by attaching two morpholine groups onto a molecule of pivalic acid. It was characterized as having an isoelectric point pI = 6.5 and exhibiting satisfactory buffering capacity at the pI. Solutions of BMMPA are transparent down to the low-UV spectral region, thus making it a potentially suitable buffer for a number of separation methods. Its use in capillary electrophoresis was demonstrated in a separation system for indirect photometric detection of anions based on an electrolyte with the anionic dye Orange G as the indirect detection probe and using BMMPA as a buffer. The use of an isoelectric buffering compound brings the advantages of a buffered electrolyte without the concomitant introduction of co-ions that would be detrimental to the indirect detection process. Submicromole per liter limits of detection for a number of inorganic and small organic ions were achieved. Optimal structural properties of the isoelectric buffer with respect to its buffering properties are discussed.

Design and performance of a light-emitting diode detector compatible with a commercial capillary electrophoresis instrument.

Indirect photometric detection in capillary electrophoresis (CE) has been predominantly performed in the UV region, in part due to a lack of suitable high-intensity and low-noise light sources in the visible spectral region. A new photometric detector based on light-emitting diodes (LEDs) as light sources and compatible with a commercially available CE instrument has been designed and constructed and its performance evaluated. The utility of this detector was successfully demonstrated by the indirect photometric detection of anions using a dye as probe and absorbance measured in the visible region. The detector exhibited very low baseline noise (around 0.03 mAU), stable output, and improved upper limit of detection linearity (502 mAU) compared with previously used LED detectors. The detector was tested for indirect detection of anions separated with an electrolyte containing 4 mM Orange G as the indirect detection probe, 10 mM histidine as an isoelectric buffer, and 0.05% hydroxypropylmethylcellulose to suppress the electroosmotic flow. Extremely low detection limits were obtained ranging from 0.16-0.36 microM (excluding chloride 0.56 microM), with separation efficiencies in the range of 154,000-274,000 theoretical plates.

General experimental aspects for capillary electrophoretic analysis of gliadins in Triticum species

Capillary electrophoresis (CE) of gliadins from Triticum species in acidic or isoelectric buffers is currently used. The performance of five buffers are compared here. The highest resolving power and the shortest time of analysis resulted associated to the isoelectric buffers. The slight different performance of the iminodiacetic acid (IDA) and aspartic acid (Asp) buffer systems should be taken into consideration before to start CE analyses. IDA buffer should be preferred when screening large germplasm collections or when a detailed characterisation of ω-gliadins is required. Asp buffer proved to be most satisfactory for cultivar discrimination or varietal identification.

Prediction of wheat dough W and P/L inflation test parameters by capillary zone electrophoresis of a protein extract followed by multivariate regression.

A procedure for the evaluation of the wheat flour hardness, based on capillary electrophoresis of a protein extract in an isoelectric acidic buffer, was developed. The 13 flour samples were extracted twice, and two injections of each extract were made. Separations were performed in a background electrolyte (BGE) containing 40 mM aspartic acid, 6 M urea, and 0.5% hydroxyethylcellulose at 60 degrees C. Using the normalized and corrected areas of 79 peaks and peak groups, a partial least squares regression (PLS1) model was able to predict the flour strength or dough deformation work (W) and the dough tenacity/extensibility ratio (P/L) (Alveograph parameters) with an average relative standard deviation in the predictions of +/- 3% and +/-8%, respectively. These values amounted to a +/- 6-8% and +/- 11% with multiple linear regression (MLR) and PLS1 models constructed by measuring only 12 peaks and peak group areas on the electropherograms.

Capillary Electrophoresis of Gliadins as a Tool in the Discrimination and Characterization of Hulled Wheats (Triticum dicoccon Schrank and T. spelta L.)

ABSTRACTTwenty‐two lines of emmer (T. dicoccon Schrank) and 10 of spelt (T. spelta L.) were analyzed using capillary electrophoresis for their gliadins. These proteins were separated on an uncoated fused‐silica capillary (30 cm long, 22 cm to detector, 50 μm i.d.) using the isoelectric buffer 40 mM aspartic acid, 4M urea, 0.5% (w/v) HEC, and 20% (v/v) acetonitrile. Samples were run for 20 min at 22kV and 42°C. By using these conditions, gliadins were separated into 21–30 components (peaks and shoulders). The major peaks eluted between 4.5 and 8.5 min. Electrophoregrams of tested lines showed qualitative and quantitative differences, including number of peaks, presence or absence of some major peaks, and areas of peaks. Lines belonging to the same species can be discriminated mainly on the basis of β‐ and ω‐gliadin patterns. The γ‐and ω‐gliadins seem to be more useful in the differentiation of emmer from spelt. The comparison of electrophoregrams relative to hulled and unhulled species evidenced the high similarity between species with the same genome composition (durum wheat‐emmer, and common wheat‐spelt).

Sensitive indirect photometric detection of inorganic and small organic anions by capillary electrophoresis using Orange G as a probe ion.

This study addresses the two major problems in the use of dyes as highly absorbing probes for indirect photometric detection in capillary electrophoresis (CE). First, effective electroosmotic flow (EOF) modification or suppression to allow separation and detection of a wide mobility range of analytes is not straightforward when electrolytes containing increased dye concentrations are used. The suppression of EOF to less than + 5x10(-9) m(2)V(-1)s(-1) was achieved with a combination of a poly(ethylenimine) (PEI)-coated capillary and the addition of the neutral polymer hydroxypropylmethylcellulose (HPMC) to the background electrolyte. Second, the deterioration of baselines due to adsorption of the dye probe to the capillary wall is generally a problem. In this work, baseline quality at higher probe concentrations was significantly improved by a rather unusual but highly effective combination of a simultaneous application of a slight overpressure (25 mbar) at the injection end during the separation, and the use of a relatively narrow capillary of 50 microm inner diameter. Both measures would appear to be counterproductive. Optimisation of the probe concentration with regard to signal-to-noise ratio resulted in an electrolyte of 4 mM Orange G, 0.05% HPMC buffered at pH 7.7 by the addition of 10.0 mM histidine isoelectric buffer. Very high separation efficiencies of 128 000-297 000 plates were made possible by the relatively high probe concentration. Combined with excellent detection sensitivity, even with the introduction of hydrodynamic flow and a reduced optical path length, these measures resulted in limits of detection ranging from 0.216 to 0.912 microM with a deuterium lamp light source (248 nm) and from 0.147 to 0.834 microM with a 476 nm blue light-emitting diode (LED) light source. Reproducibility over 30 consecutive runs without changing the electrolyte was excellent, with relative standard deviation (RSD) values of 0.14-0.80% for migration time, 1.27-3.36% for peak area and 0.88-5.12% for peak heights. The optimised electrolyte was used for the analysis of inorganic anions in air filter samples, providing good agreement with results obtained by ion chromatography.

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest.

Capillary electrophoresis (CE) is an automated approach to electrokinetic separations that has had a deep impact in all fields of life sciences, including biomedical and biotechnological research and clinical and forensic practice. The present review highlights aspects of peptides and proteins separations, with particular emphasis on macromolecular analytes of biomedical interest. Among the various CE techniques available, a novel methodology is here illustrated consisting in separations in acidic, isoelectric buffers, which have the advantage of protonating the silica wall, thus minimizing interactions of proteinaceous material with the siliceous surface, while allowing delivery of high voltage gradients, due to their low conductivities. The review ends with applications of CE to the analysis of folding/unfolding/refolding/misfolding of proteins, a field which has deep implications in the biomedical arena, since it is connected to a host of disorders, such as prion protein diseases.