Analysis of bovine whey proteins in soybean dairy-like products by capillary electrophoresis

The simultaneous separation of bovine whey proteins [alpha-lactalbumin and beta-lactoglobulin (A+B)] and soybean proteins was performed, for the first time, by capillary electrophoresis. Different experimental conditions were tested. The most suitable consisted of 0.050 M phosphate buffer (pH 8) with 1 M urea and 1.2 mg/ml methylhydroxyethylcellulose, UV detection at 280 nm, 15 kV applied voltage, and 30 degrees C temperature. Quantitation of bovine whey proteins in a commercial powdered soybean milk manufactured by adding bovine whey to its formulation was performed using the calibration method of the external standard. Direct injection of a solution of the powdered soybean milk only enabled quantitation of alpha-lactalbumin in the commercial sample. Detection of beta-lactoglobulin (A+B) required acid precipitation of the solution of the sample in order to concentrate bovine whey proteins in the supernatant prior to the analysis of this protein in the whey obtained. Since alpha-lactalbumin could also be quantitated from the injection of the whey, the simultaneous determination of alpha-lactalbumin and beta-lactoglobulin (A+B) was possible upon acid precipitation of the powdered soybean milk solution. Detection limits obtained were 14 microg/g sol. for alpha-lactalbumin and 52 microg/g sol. for beta-lactoglobulin (A+B) which represent protein concentrations about 60 microg/100 g sample for alpha-lactalbumin and 100 microg/100 g sample for beta-lactoglobulin (A+B).     

Analysis of major milk whey proteins by immunoaffinity capillary electrophoresis coupled with MALDI-MS

Two major milk whey proteins, β-lactoglobulin and α-lactalbumin, are among the main cow milk allergens and can cause allergy even at a very low concentrations. Therefore, these proteins are interesting targets in food analysis, not only for food quality control but also for highlighting the presence of allergens. Herein, a sensitive analysis for β-lactoglobulin and α-lactalbumin was developed using immunoaffinity capillary electrophoresis hyphenated with MALDI-MS. Magnetic beads functionalized with appropriate antibodies were used for β-lactoglobulin and α-lactalbumin immunocapture inside the capillary. After elution from the beads, analyte focusing and separation were performed by transient isotachophoresis followed by MALDI-MS analysis performed through an automated iontophoretic fraction collection interface. A LOD in the low nanomolar range was attained for both whey proteins. The method developed was further applied to the analysis of different milk samples including fortified soy milk.     

Whole-column imaging capillary electrophoresis of proteins with a short capillary

Whole-column imaging capillary electrophoresis with a short capillary is discussed. A short capillary (3-6 cm) coated with either fluorocarbon or polyacrylamide was used as a separation capillary. The whole capillary was illuminated with 280 nm light, and the transmitted light was monitored by a linear charge-coupled device (CCD). For the short capillary, hydrodynamic flow caused by a subtle height difference between the anodic and cathodic reservoirs affected the sample migration in the capillary greatly. Several sample injection methods, including use of a cross connection, sealing of the capillary ends with a gel, and use of a gel-filled capillary, have been discussed. The experimental results showed that the peak height decreased and peak width increased with the electromigration distance. Therefore, higher sensitivity was obtained in a short capillary rather than a long capillary. The whole-column imaging CE with the short capillary has been applied for the study of conjugation reactions of protein cytochrome c with sodium dodecyl sulfate (SDS) and the dye Congo Red. The method has also been used for in situ monitoring of the electrophoretic protein desorption process. Our technique is a unique tool for the study of protein binding reactions and the interaction between analyte and inner wall of the capillary.     

两种表征核酸适配体-靶蛋白亲和作用的毛细管电泳方法比较

适配体-靶分子间的亲和作用表征是理解和应用核酸适配体发挥特异亲和作用的基本前提,CE技术则为上述表征提供了多模式的简捷途径,但多种模式体系间的结果往往存在差异,导致CE亲和评价可靠性和进一步应用受到限制,亟须建立多CE方法测定适配体-靶分子间亲和作用的系统比较研究。该研究以凝血酶及其特异性作用于肝素结合位点的适配体29mer为模型体系,基于CE-激光诱导荧光检测,引入CE-迎头分析(FA)评价方法,并比较其与预平衡-毛细管区带电泳(PE-CZE)的异同。首先进行了CE-FA方法分离条件的优化,37℃、0.5 h孵育完全后进样,进样时间为30 s,在较低工作温度(15℃)、较短毛细管长度(30 cm)及生物相容性好的缓冲体系2×TG(Tris-甘氨酸缓冲液,pH 8.5)条件下,经15 kV分离时,得到了稳定的荧光标记29mer(F29mer)-凝血酶复合物及游离F29mer平台峰。加入1 g/L牛血清白蛋白(BSA),有效提高了CE-FA平台峰高及迁移时间的重复性。详细讨论了两种方法下6种拟合方式的结果及特点。针对CE-FA和PE-CZE法,以结合适配体/游离适配体的浓度比对游离适配体...     

Electroosmotic pump-assisted capillary electrophoresis of proteins

A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated.     

Separation of selected metal-binding proteins with capillary zone electrophoresis

Several metal-binding proteins, including albumin, carbonic anhydrase, conalbumin, cytochrome c, ferritin, hemoglobin, myoglobin, plasma amine oxidase, superoxide dismutase and transferrin were separated with capillary zone electrophoresis (CZE) in uncoated and coated capillaries. Phosphate and tetraborate buffers achieved complementary separation selectivities. Optimised pre-wash protocols for uncoated capillaries using 0.1 M HCl as a rinsing solution for the borate buffer and a combination of 0.1 M NaOH and 0.1 M HCl for the phosphate system improved the stability of migration times considerably with coefficients of variation between 0.10 and 0.77% (n=7) instead of up to 2.92% with inappropriate rinsing conditions. Capillaries coated with poly(vinyl alcohol) and equipped with a 150 μm i.d. bubble cell increased the signal-to-noise ratio by a factor three, additionally improving the resolution. For commercial protein standards, which gave several peaks in CZE with UV detection, MS data proved the presence of proteinaceous contaminants. Molecular weights (M_r) of proteins experimentally determined from MS data showed deviations from theoretical M_r as small as 0.002-0.021%. Applicability of the developed separation for clinical samples is shown for human serum.     

On-line preconcentration methods for capillary electrophoresis

The limits of detection (LOD) for capillary electrophoresis (CE) are constrained by the dimensions of the capillary. For example, the small volume of the capillary limits the total volume of sample that can be injected into the capillary. In addition, the reduced pathlength hinders common optical detection methods such as UV detection. Many different techniques have been developed to improve the LOD for CE. In general these techniques are designed to compress analyte bands within the capillary, thereby increasing the volume of sample that can be injected without loss of CE efficiency. This on-line sample preconcentration, generally referred to as stacking, is based on either the manipulation of differences in the electrophoretic mobility of analytes at the boundary of two buffers with differing resistivities or the partitioning of analytes into a stationary or pseudostationary phase. This article will discuss a number of different techniques, including field-amplified sample stacking, large-volume sample stacking, pH-mediated sample stacking, on-column isotachophoresis, chromatographic preconcentration, sample stacking for micellar electrokinetic chromatography, and sweeping.     

High-performance capillary electrophoresis of hydrophobic membrane proteins

Hydrophobic membrane proteins, extrinsic and intrinsic ones, were separated by high-performance capillary zone electrophoresis (HPCZE) and high-performance capillary isotachophoresis (HPCITP). In the case of HPCZE with both coated and uncoated quartz capillaries the addition of 7 M urea to the separation buffers was necessary to achieve reproducible results. In the HPCITP experiments PTFE capillaries were used. When spacers were used, e.g., ampholytes, additional splitting of peaks was observed. The splitting was caused by the microheterogeneity of the investigated proteins, which are differently glycosylated and/or phosphorylated.     

Isoelectric focusing of proteins in capillary electrophoresis with pressure-driven mobilization

Chromatographia - An isoelectric focusing (IEF) method in the capillary format with wide linear pH range (pH 3–10) and high resolution has been developed for separations of proteins. The...     

Development of a method for quantitative analysis of the major whey proteins by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

The main whey proteins have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus provided with a laser-induced fluorescence detector. Several parameters controlling on-capillary derivatization of proteins, including pH, mixing time, reaction time, concentration of the reagents (potassium cyanide and FQ), and reaction temperature, were optimized. Coefficient variations were lower than 1% for migration time and 7% for peak height. Assay detection limits for the different proteins were in the range 5 nM to 10 nM. The method developed was applied to the separation of the major whey proteins in a laboratory-made cheese whey and in an infant food formulated with milk. In addition, the beta-LG content of these samples was quantitated. The results showed good agreement with those given by an RP-HPLC method and with those reported in the literature.     

软骨藻酸作用蛋白质的亲和毛细管电泳筛选

基于亲和毛细管电泳,定性比较了血浆、肠道及细胞线粒体中所选的9种重要功能蛋白质与神经毒性生物毒素--软骨藻酸(domoic acid, DA)的相互作用。以DA淌度比变化量 Δ M与蛋白质浓度L作图,根据斜率值大小可比较DA与蛋白质作用的相对强弱。结果如下:可与DA相互作用的有6种蛋白质,作用强弱为:人凝血酶 > 细胞色素C≈胰蛋白酶≈免疫球蛋白E (Ig E) ≈核糖核酸酶A > λ 核酸外切酶;而铁蛋白、转铁蛋白、凝集素与DA未表现出相互作用和亲和力。实验表明,亲和毛细管电泳具有高效、快速、所需样品量低的优点,可用于DA作用靶蛋白的筛选,为DA的毒性机制研究和毒性防护提供基础信息。     

Optimization methods in chromatography and capillary electrophoresis

Many methods have been developed in order to optimize the parameters of interest in either chromatography or capillary electrophoresis. In chemometric approaches experimental measurements are performed in such a way that all factors vary together. An objective function is utilized in which the analyst introduces the desired criteria (selectivity, resolution, time of analysis). Simplex methods and overlapping resolution maps are declining. Factorial designs and central composite designs are more and more popular in electrodriven capillary separations since the number of parameters to master is much larger than in either GC or LC. The use of artificial neural networks is increasing. The advantage of chemometrics tools is that no explicit models are required, conversely the number of experiments to perform may be high and boundaries of the domain are not straightforward to draw and the approach does more than is required. When models are available optimization is easier to perform by regression methods. Computer assisted methods in RPLC are readily available and work well but are still in infancy in CE. Linear solvation energy relationships seem a very valuable tool but estimates of coefficients still require many experiments.     

Separation of β-lactoglobulin A, B and C variants of bovine whey using capillary zone electrophoresis

β-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the β-lactoglobulin A, B and C variants. Three buffer systems [borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pK_a range (pH 5.3–7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified β-lactoglobulin A and B proteins and a β-lactoglobulin AC whey. The method was then used to phenotype β-lactoglobulin in a sample population of New Zealand Jersey cows.     

Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking

The paper describes a method of size separation of proteins by capillary sieving electrophoresis with cationic surfactant. Proteins are separated within 12 min with repeatability of migration times better than 0.2%. Some proteins achieve the separation efficiency of 200,000 theoretical plates. The method can be used for determination of protein relative molecular masses. The accuracy of the determined relative molecular masses and the limitation of the method were investigated by the analysis of more than 60 proteins. The method also allows separation of protein oligomers. Proteins can be quantitated after the electrokinetic injection in the concentration range 0.07-0.43?g/L. The average detection limit is about 2?mg/L.     

Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins

The number of proteins produced by recombinant DNA technology continues to grow at a rapid pace. In this review, the emphasis is on proteins that are of therapeutic interest. Aspects of protein analysis, such as glycoform separation of proteins produced in mammalian cells and the separation of oligosaccharides for structure elucidation, are covered. The use of antibodies as therapeutic proteins is growing and currently antibodies are the largest class of proteins produced by biotechnology. This has merited a separate section on analysis of antibodies by capillary electrophoresis (CE). Applications of mass spectrometry as an ancillary technique, used in conjunction with CE, are also covered briefly. This review covers the literature since 1999.     

A preliminary evaluation of serum proteins by capillary electrophoresis

We evaluated the 5-band Serum Proteins by Capillary Electrophoresis kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.) on the BioFocus 2000 CE (Bio-Rad) against conventional agarose gel electrophoresis (AGE) (Helena Laboratories, Beaumont, TX, U.S.A.). Serum from 60 patients was initially screened by AGE and divided into three groups: 1) normal electrophoretic pattern (n = 36, mean total protein 67.7 g/L), 2) monoclonal/oligoclonal gammopathy (n = 14, mean total protein 78.8 g/L), and 3) polyclonal gammopathy (n = 10, mean total protein 77.4 g/L). These samples were concurrently analyzed on the BioFocus 2000. Intraassay and interassay CVs for the five fractions for a normal sample were 0.17-1.44% and 0.42-9.11%, respectively, and 0.21-3.37% and 0.29-3.61%, respectively, for a sample with monoclonal gammopathy. Correlation coefficients for albumin and the albumin/globulin (A/G) ratio were 0.8891 (albumin range 17-93 g/L) and 0.8276 (A/G ratio range 0.39-7.81), respectively. The A/G ratio alone could not discriminate between the three groups. Capillary zone electrophoresis (CZE) correctly identified 33 of 36 samples in the normal group, and 22 of 24 samples in the other two groups, giving a clinical sensitivity and specificity of 91.7%. Our preliminary evaluation shows that protein separation by CZE is a simple, rapid, and automated alternative to conventional AGE.     

Recent developments in high-performance capillary electrophoresis of cereal proteins

Cereal proteins play important nutritional and functional roles in human foods and are also important components of animal feeds. As such, cereals are a major economic factor around the world. Because of their importance, cereal proteins have been widely studied. A new emerging technique for studying cereal proteins is high-performance capillary electrophoresis (HPCE). This review focuses mainly on new methods and applications of HPCE to cereal proteins that have been reported in the last three years.     

Recent developments in capillary zone electrophoresis of proteins.

This review article with 125 references describes recent developments in capillary zone electrophoresis of proteins. It encompasses approximately the last two years, from the previous review (V. Dolník, Electrophoresis 1997, 18, 2353-2361) through Spring 1999. Topics covered include modeling of the electrophoretic properties of proteins, sample preconcentration and derivatization, wall coatings, improving selectivity, special detection techniques, and applications.     

Precision study on capillary electrophoresis methods for metacycline

A CE method for metacycline (MTC) determination was investigated in an inter-laboratory experiment. Many problems were encountered in this study, most of which were related to the transfer of the method to different CE equipment. The reported problems could be classified into different categories: problems related to the precision, to the parameters in the protocol, and to the MTC peak shape. As the peak shape problem was partially responsible for the poor precision, a new CE method was developed in order to obtain a good MTC peak shape on all equipment. The precision of this new method for MTC determination was examined in an intermediate precision study, where the influence of the factors "time" and "equipment" was investigated. Although the new method could be transferred to different instruments, the precision remained poor mainly due to the contributions of the between-replicate and the between-injection variances.