Capillary electrophoresis separation of high molecular weight glutenin subunits in bread wheat (Triticum aestivum L.) and related species with phosphate-based buffers

This study focused on optimizing phosphate-based buffers and other capillary electrophoresis (CE) parameters for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), emmer (Triticum dicoccum, AABB, 2n = 4x = 28) and Aegilops tauschii (DD, 2n = 2x = 14). The fast and high-resolution separation of HMW-GS was achieved using 0.1 M phosphate-glycine buffer (pH 2.5, containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose) at 12.5 kV and 40 degrees C with 25 microm inside diameter (ID)x27 cm uncoated fused-silica capillary. In general, one sample separation can be analyzed in 15 min. The good run-to-run repeatable separation of HMW-GS could be obtained with a relative standard deviation of less than 1% when capillaries were rinsed with 1 M phosphoric acid for 2 min, followed by separation buffer for 2 min after each separation. The HMW-GS from some bread wheat cultivars as well as tetraploid and diploid accessions was separated by the CE method described above, and all subunits detected were well characterized and readily identified. Some HMW-GS showed reversed mobilities and elution order compared to the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-CE. Particularly, most of the HMW-GS analyzed with the CE buffer used were separated into multiple peaks, generally a high peak plus a minor peak. CE appears to be capable of separating and characterizing HMW-GS with fast and high-resolution features, therefore it is expected to be useful for specific germplasm screening and desirable HMW-GS identification in wheat quality improvement.     

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis

Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I-V) characteristic and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes-fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters.     

Electrochemical determination of hesperidin using mesoporous SiO2 modified electrode

A simple micro-capillary electrophoresis system to be used as disposable device was developed. A short commercial capillary was used as the separation channel, hydrostatic pressure generated by the sample employed for injection, and a voltage of 200 V used for separation in a 6 cm long capillary assisted by hydrostatic pressure of the carrier. The device was used for the separation of dopamine and catechol. Good reproducibility and efficiency was obtained. Because the instrumentation and operation conditions were simplified, and a replaceable modular separation channel was used, the proposed micro-capillary electrophoresis system is potentially useful in disposable devices.     

Precision of micellar electrokinetic capillary chromatography in the determination of seven antibiotics in pharmaceuticals and feedstuffs

Validation of analytical procedures is important for their efficient and reliable application. The International Conference on Harmonisation (ICH), Food and Drug Administration (FDA) and pharmacopoeia guidelines achieved a great deal in harmonising the definitions of the required validation characteristics. It is well known that poor reproducibility limits the practical implementation of capillary electrophoresis (CE). A precision study on four different MEKC methods was performed with 11 samples, containing seven antibiotics, by two analysts, in few days, on two capillary electrophoresis instruments. Five pharmaceutical preparations and three animal feeds were used. Precision was statistically analysed using migration time, peak area and height of each compound, as well as electroosmotic front (EOF). In 25 of 31 cases, the reproducibility of peak area, peak height and migration time was good (<5%). In most cases the reproducibility of peak area was much better than the reproducibility of peak height. The worst reproducibility that we observed was 12.7% for peak height and 7.6% for peak area.     

Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker’s Respiratory Allergy

Wheat allergens are responsible for symptoms in 60–70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21–27 kDa that were recognized by the pooled baker’s IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.     

糖类的毛细管电泳及芯片毛细管电泳

 糖类化合物在生物体内发挥多方面的作用。糖研究的复杂性在于其结构的复杂多变。高效毛细管电泳作为一种快速、高效的分离分析手段已广泛应用于糖的研究。芯片毛细管电泳是近几年来发展起来的新的分析技术 ,并已经在生命科学的研究中得到较广泛的应用。就各种糖类化合物的毛细管电泳的分析策略、检测条件及糖类化合物的芯片毛细管电泳进行了阐述 ,共 4 8篇。     

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography

A collaborative study on the robustness and portability of a capillary electrophoresis‐mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis‐mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath‐flow capillary electrophoresis–mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin‐digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3–12 and 9–29% RSD, respectively. These results demonstrate that capillary electrophoresis‐mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis‐mass spectrometry applications in biopharmaceutical analysis and related fields.     

Matrix-assisted laser desorption/ionization time-of-flight based wheat gliadin protein peaks are useful molecular markers for wheat genetic study

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation has been used to analyze wheat seed gliadins as an alternative to other established methods, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), etc. The MALDI-TOF approach has shown to have many advantages such as high resolution, cost effectiveness and high throughput. MALDI-TOF-based gliadin profiles have been used for fast wheat cultivar identification. However, the genetic information represented by individual gliadin peaks has not been utilized. In this study a wheat doubled haploid population with a genetic linkage map of good coverage was used to assay individual gliadin peaks from MALDI-TOF profiles as molecular markers. Eight segregating peaks in the population were scored as polymorphic across the population. The 1 to 1 segregating ratios validated the scoring of the peaks and all peaks were mapped to the expected chromosomes or linkage groups on the available linkage map: 1 peak on chromosome 1A, 1 on 6A, 4 on 6B and 2 on 6D.     

Development of a capillary electrophoresis system coupled to sequential injection analysis and evaluation by the analysis of nitrophenols

A combination of a laboratory-made capillary electrophoresis system and a sequential injection analysis equipment is described. For characterization, the system was successfully applied to the separation and quantification of nitrophenols. A blue LED was used as light source, and hydrodynamic injection was carried out by using a pressure-stable solenoid valve and an inflatable pressure reservoir. A good reproducibility of migration time (0.5%) and peak heights (5%) were obtained. The calibration by using peak heights was found to be linear up to 776?µmol?L^?1 for all three compounds. The system was robust and reliable for autonomous analysis without observation. All maintenance requirements including the conditioning of the capillary and flushing of both buffer reservoirs were carried out automatically. Instrumentation aspects of the capillary electrophoresis part are compared with former described hyphenated flow systems showing maximal operation versatility. Instrumental control and data evaluation were carried out using the software package AutoAnalysis.     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Rational approach to quantitative sodium dodecyl sulfate capillary gel electrophoresis of monoclonal antibodies

Sodium dodecyl sulfate capillary gel electrophoresis has been used to separate and quantify murine monoclonal antibodies. The method uses a murine IgG, whose subclass differs from the analyte antibody, as an internal reference. The internal reference is chosen based on knowing that mouse IgG1 can be separated from mouse IgG2a or IgG2b. Good intra- and inter-day reproducibility [relative standard deviation (RSD)<2%] of peak-area ratio has been obtained. A calibration curve also demonstrates high linearity (R2=0.9999) of response for the analyte. The described method is highly suitable for accurate determination of the antibody concentration even if a capillary electrophoresis apparatus is unable to provide good injection reproducibility.     

Capillary electrophoresis of polycationic poly(amidoamine) dendrimers

Generation 2 to generation 5 poly(amidoamine) (PAMAM) dendrimers having different terminal functionalities were analyzed by capillary electrophoresis (CE). Polyacrylamide gel electrophoresis was also used to assess the composition of the individual generations for comparison with the CE results. Separation of PAMAMs can be accomplished by either using uncoated silica or silanized silica capillaries, although reproducibility is poor using the uncoated silica capillary. To improve run-to-run reproducibility, silanized capillary was used and various internal standards were also tested. Relative and normalized migration times of primary amine terminated PAMAM dendrimers were then determined using 2,3-diaminopyridine (2,3-DAP) as an internal standard. Using silanized capillaries and internal standards, the relative and normalized migration times are fully reproducible and comparable between runs. Apparent dimensionless electrophoretic mobilities were determined and the results were compared to theoretical calculations. It is concluded that for PAMAMs a complex separation mechanism has to be considered in CE, where the movement of the ions is due to the electric field, but the separation is rather the consequence of the adsorption/desorption equilibria on the capillary wall ("electrokinetic capillary chromatography"). The described method may be used for quality control and may serve as an effective technique to analyze polycationic PAMAM dendrimers and their derivatives with different surface modifications.     

Intra- and interinstrument reproducibility of migration parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

The intra- and interinstrument reproducibilities of four capillary electrophoresis instruments were studied for identification purposes in systematic toxicological analysis (STA). A test set of 20 acidic test compounds and 5 reference compounds were analyzed for five days on each instrument using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The buffers consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate and 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries at an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA. To deal with the poor reproducibility of the migration time, we recently introduced the corrected effective mobility. In this study, we investigated the intra- and interinstrument reproducibility of the migration time, the effective mobility, and the corrected effective mobility. Large differences in intra-instrument reproducibility were found when the migration time was used. The calculation of the effective mobility and the corrected effective mobility diminished these differences and enhanced the interinstrument reproducibility roughly by a factor 3. For (corrected) effective mobilities, intrainstrument reproducibilities were between 0.8-2.6% and interinstrument reproducibilities were between 3.2-3.9%.     

Direct assay of glutathione peroxidase activity using high-performance capillary electrophoresis.

A fast, sensitive and direct method has been developed for the determination of glutathione peroxidase activity (both selenium- and non-selenium-dependent) in cell-free preparations. The assay is based on the separation and quantitation of reduced and oxidized glutathione by capillary electrophoresis. The electrophoretic separation buffer was 100 mM sodium tetraborate (pH 8.2) containing 100 mM sodium dodecylsulphate. A micellar electrokinetic mechanism took place under these conditions, and a total mass recovery was observed for both peptides. The reproducibility of migration times was excellent (less than 3% variability). A linear detector response range was observed in the range 5-50 U/ml, and both the reproducibility and accuracy were satisfied. Samples out of this linear range could be analysed by either increasing the reaction time or diluting the enzyme preparation. The results obtained with the new direct capillary electrophoresis assay were compared with those derived from a reversed phase high-performance liquid chromatographic and spectrophotometric coupled assay. A very good agreement was found between the two direct assay methods in all samples. Capillary electrophoresis is a versatile technique that allows the automation of the glutathione peroxidase assay in a reproducible manner and within a relatively short time with sufficient accuracy and precision.     

Interlaboratory reproducibility of mobility parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

An interlaboratory pilot study was performed to determine the reproducibility of mobility parameters in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The study was performed by an intended small number of laboratories (three) that used different brands of instruments (two). The effective mobility was corrected using standards by a method that was recently introduced to obtain a more reproducible migration parameter. A test set of 20 acidic test compounds and 5 reference compounds were analyzed during five days in each laboratory using CZE and MEKC. Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). Analyses were carried out using fused-silica capillaries at an electric field strength of either 52.6 kV/m or 37.5 kV/m. The interlaboratory reproducibility (mean RSD) of the effective mobility was 3.0% for CZE and 6.7% for MEKC. After applying the correction method, these values became 3.0% for CZE and 3.3% for MEKC, which is adequate for systematic toxicological analysis (STA) applications. A significant improvement of reproducibility for the calculated corrected effective mobility mu(eff)c was observed when variations are high. Therefore, it is recommended to use the correction method in interlaboratory situations, especially when instruments and capillaries from different manufacturers are used.     

A New Capillary Electrophoresis Apparatus with Piezoelectric Ceramics High Voltage Source and Amperometric Detector

IntroductionHighperformancecapiliar}'electrophoresis(HPCE)isanewanalyticaltechnology'rapidly'developedinrecentyears.Withtheadvantagesofsmallsample.highsensitivity,highresolution.rapidanalysisandverycheaprunning,ithasbeenappliedinchemistry'.lifescienc...     

聚丙烯酰胺电泳法测定小麦种子纯度方法的改进

使用SE-2000A型对小麦、玉米专用的电泳仪，对GB/T3543.5-1995聚丙烯酰胺电泳法测定小麦种子纯度方法的样品提取、点样量及电泳条件进行了改进，结果表明：改进后使麦醇蛋白溶蛋白电泳图谱的蛋白质区带界限更明显，公共带和特征带清晰、可靠，且样品提取时间缩短了23.5h。     

A capillary electrophoresis chip with hydrodynamic sample injection for measurements from a continuous sample flow

A microchip-based capillary electrophoresis device supported by a microfluidic network made of poly(dimethylsiloxane), used for measuring target analytes from a continuous sample flow, is presented. The microsystem was fabricated by means of replica molding in combination with standard microfabrication technologies, resulting in microfluidic components and an electrochemical detector. A new hydrodynamic sample injection procedure is introduced, and the maximum number of consecutive measurements that can be made with a poly(dimethylsiloxane) capillary electrophoresis chip with amperometric detection is investigated with respect to reproducibility. The device features a high degree of functional integration, so the benefits associated with miniaturized analysis systems apply to it.     

Microchip Electrophoretic Analysis of Phaseolin Patterns and Its Comparison with Currently Used SDS-PAGE Techniques

The goal of this work was to compare reproducibility of phaseolin patterns of common bean obtained by two electrophoretic protein separation techniques including the conventional SDS-PAGE and an automated chip electrophoresis system. Five standard cultivars of common bean provided by the United States Department of Agriculture (Beltsville, Maryland) that represented five phaseolin types, T (Tendergreen), C (Contender) and S (Sanilac), B (Boyaca) and P (Pampa), were used in this study. Comparison of the phaseolin patterns revealed that the chip-on-a-lab electrophoresis provided a good reproducibility. The phaseolin polymorphism included four to seven polypeptides typical for the pattern composition of the T, C and S types. The polymorphism of the B and P patterns was also established. Phaseolin polypeptides separated by the microchip electrophoresis exhibited differences with respect to the molecular weights and electrophoretic mobility as compared to the SDS-PAGE technique. This phenomenon could be attributed to the absence of a solid separation phase in the microchip electrophoresis. Moreover, this technique has potential to substantially accelerate screening of large bean germplasm collections since it allows for the accurate analysis of the higher number of individual plants within accessions than the conventional, tedious and time consuming SDS-PAGE method.     

微波消解-电感耦合等离子体质谱(ICP-MS)法测定山东小麦中铬、镍、铜、砷、镉、铅、锌的含量

建立了微波消解-电感耦合等离子体质谱(ICP-MS)法测定山东小麦中7种重金属元素(Cr、Ni、Cu、As、Cd、Pb、Zn)的方法,并对山东地区千余份小麦进行分析。结果表明,通过使用适宜的内标和He碰撞模式,7种重金属元素的方法检出限在0.0007~0.09 mg/kg,相对标准偏差(RSD,n=6)在10%以内,标准曲线线性关系良好,相关系数均大于0.999,方法用于国家有证标准物质(GSB-3、GSB-4、GSB24)的测定,结果与标准值相符。方法灵敏度高,重现性好,定量准确,可用于大批量小麦样品的测定。