Acetonitrile as a buffer additive for free zone capillary electrophoresis separation and characterization of maize (Zeamays L. ) and sorghum (Sorghum bicolor L. Moench) storage proteins

An improved method for separating and characterizing maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) storage proteins by free zone capillary electrophoresis (FZCE) was developed. Previous electrophoretic methods for analyzing these proteins required high concentrations of urea to maintain protein solubility during separation. To overcome disadvantages of urea, we developed a FZCE method that mimicked reversed-phase high-performance liquid chromatography (RP-HPLC) in that it used high levels of acetonitrile (ACN) at low pH. The optimized FZCE buffer system consisted of 80 mM phosphate-glycine buffer, nominal pH 2.5, containing 60% ACN and a cellulose derivative to dynamically coat capillary walls. Resolution was similar to or higher than that previously achieved by FZCE buffers utilizing 8 M urea as a buffer additive. ACN concentrations of at least 50% were necessary to achieve acceptable separations; this ACN concentration is approximately that necessary to extract these storage proteins. ACN was equally effective as traditional ethanol solvents and 8 M urea for solubilizing maize and sorghum proteins. The ACN-based FZCE buffer system gave high repeatability (<0.3% relative standard deviation, measured over 15 consecutive injections) for migration time. Subclasses of maize and sorghum storage proteins were identified, and genotypes of each cereal were successfully differentiated using ACN-containing buffers. This FZCE method may be applicable for the analysis of other hydrophobic proteins without the use of urea.     

Investigation and optimization of the factors influencing sorghum protein extraction

To optimize the extraction of sorghum proteins, several variables were examined: sample-to-solvent ratio, detergent type and concentration, reducing agent type and concentration, extraction time, and buffer pH and concentration. Samples were quantified and characterized by RP-HPLC, FZCE, and nitrogen analysis. These studies revealed that pH, detergent type, reducing agent type, and sample-to-solvent ratio all had significant effects on the levels of protein extracted. Increasing SDS concentration (2%) and solvent-to-flour ratio (20:1) with multiple 5 min extracts reduced extraction time by 35-80% while still extracting the same levels of total protein relative to the control methodology. Reproducibility using the multiple extractions was found to be excellent with relative standard deviations of <2% for consecutive extractions.     

Separation of Water‐Soluble Proteins from Cereals by High‐Performance Capillary Electrophoresis (HPCE)

Most research concerning grain proteins has concentrated on the gluten storage proteins. The albumins and globulins are the water‐ and salt‐soluble proteins that contain biologically active enzymes and enzyme inhibitors. A free‐zone capillary electrophoresis method was developed to separate these proteins. Optimization included sample extraction method, capillary temperature, buffer composition, and additives. The optimal conditions for separation of these proteins was 50 μm i.d. × 27 cm (20 cm to detector) capillary at 10 kV (with a 0.17 min ramp‐up time) and 25°C. The optimum buffer was 50 mM sodium phosphate, pH 2.5 + 20% acetonitrile (v/v) (ACN) + 0.05% (w/v) hydroxypropylmethyl‐cellulose (HPMC) + 50 mM hexane sulfonic acid (HSA). Sample stability was an issue that was addressed by lyophilizing fresh extracts and redissolving in aqueous 50% ethylene glycol and 10% separation buffer. This method was successfully used in both wheat flour and whole meal samples. Comparisons were made of several wheats of different classes as well as several cereal grains. This methodology could be useful in screening cereal grains for important enzymes and their impact on end‐use quality such as food functionality, food coloration, and malting quality.     

Free-zone capillary electrophoresis analysis of hordein patterns at different stages of barley malting

We have carried out a comparison of hordein patterns at different stages of the malting process using free-zone capillary electrophoresis (FZCE). FZCE has proved to be a suitable technique for the separation and characterization of hordeins in barley seeds. Assays of protein extraction and electrophoretic procedures led us to conclude that hordeins were best extracted with 40% ethanol and analyzed using 50 mM phosphate-glycine, pH 2.5, containing 20% ACN and 0.05% HPMC, at 12.5 kV and 45 degrees C, with 10 s hydrodynamic injection at 0.5 psi and 50 microm i.d. x 31 cm uncoated fused-silica capillary. Our results afford useful information about changes in the composition of these proteins in barley during malting.     

A Rapid Protein Digestibility Assay for Identifying Highly Digestible Sorghum Lines

Protein digestibility in sorghum (Sorghum bicolor (L.) Moench) lines was determined using two standard procedures (pepsin digestibility and pH‐stat) and compared with a newly developed, rapid electrophoresis‐based screening assay. The new assay was based on the rate of α‐kafirin disappearance after pepsin digestion. α‐Kafirin, the major sorghum storage protein, makes up ≈60–70% of the total protein in the grain. In the new assay, samples were first digested with pepsin for 1 hr, and undigested proteins were then analyzed by SDS‐PAGE. The intensitizes of the undigested α‐kafirin bands were measured. Higher band intensity indicated lower protein digestibility. The new assay was significantly correlated with the standard pepsin digestibility assay (r = −0.96, n = 16) after which it was patterned. The same was true of the pH‐stat procedure (r = −0.85, n = 16). This implies that the new assay is comparable to existing procedures and can be used for screening sorghum lines for protein digestibility. Two groups consisting of high‐protein digestibility and wild‐type sorghum lines were identified when the new assay was tested on 48 sorghum lines derived from crosses of wild‐type and mutant high protein digestibility lines, indicating that the new assay was efficient in differentiating between the two groups. Advantages of the new assay over the standard procedures include considerable reduction in analysis time and sample size required for the analysis. For example, analysis time was reduced by 20% and sample size by 10% when the new assay was used as compared with the pH‐stat procedure. We estimate that ≈60 sorghum lines can be screened in a day by a single operator using the new assay.     

Effect of Aelia spp. and Eurygaster spp. Damage on Wheat Proteins

The effect of Aelia spp. and Eurygaster spp. wheat bugs on the protein fractions of different wheat cultivars has been studied by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and free‐zone capillary electrophoresis (FZCE). Those methods were used to quantify and characterize the extent of protein modification. A decrease in the amount of alcohol‐insoluble polymeric proteins along with an increase in the alcohol‐soluble polymeric proteins and gliadins were observed in damaged wheat. The high molecular weight (HMW) and low molecular weight (LMW) glutenin fractions were barely detected in the incubated damaged wheat from some cultivars, which indicated hydrolysis of those proteins by the bug proteinases. In damaged wheats, both incubated and unincubated, gliadin electrophoregrams revealed the presence of some new peaks with mobilities similar to the ω gliadins. The overall results suggest that the bug proteinases are potent enzymes that appear to be nonspecific because they hydrolyze all gluten proteins.     

Application of capillary electrophoresis on the characterization of cadmium‐induced polypeptides in roots of pea plants

Capillary electrophoresis (CE) has revealed as a powerful technique for a rapid and effective evaluation of the synthesis of polypeptides induced by cadmium (Cd) in root tissues of Cd‐resistant pea plant. CE was performed on root extracts in capillary gel electrophoresis (CGE) using 3% PEG 2November 1998 in Tris‐Tricine buffer as a dynamic sieving polymer solution and in free solution (FSCE) with 0.1M Tris‐Tricine at pH 8.3 as running buffer. By comparison with control, CGE electropherograms of root extracts of Cd‐resistantpea showed a new peak, characteristic of newly synthesized polypeptidic fraction with apparent molecular weight of 10 kDa. In order to get more information, we characterized further the new synthesized polypeptidic fraction as charge to size ratios. Results obtained by FSCE in free solutions showed that polypeptidic fraction split into several components with different charge to size ratios, very close to that of glutathione, the starting molecule for the biosynthesis of phytochelatins. In addition, results suggested a co‐occurrence of MT‐like proteins at low molecular weight and phytochelatins.     

Detection of smoked paprika "Pimentón de La Vera" adulteration by free zone capillary electrophoresis (FZCE)

The purpose of this work was to develop a procedure based on protein analysis by free zone capillary electrophoresis (FZCE) that can be used in the determination of smoked paprika "Pimentón de La Vera" adulteration with paprika elaborated from varieties of pepper foreign to the "La Vera" region, in central western Spain. Two autochthonous varieties of pepper, Jaranda and Bola, and the variety Papri Queen, foreign to the "La Vera" region, were used in the study. Several aqueous solutions for solubilization of the methanol-soluble proteins were tested, and the FZCE conditions of capillary dimensions, FZCE buffer concentrations, and detection wavelengths were optimized. On the basis of the results, 30% (v/v) acetonitrile was adopted as the suspending solution for routine analysis, and the optimal FZCE parameters were 75 microm inner diameter and 57 cm total length capillaries, 8.75 mM phosphate/20.6 mM tetraborate as run buffer, and 256 nm as detection wavelength. This method was found to give excellent repeatability of the corrected migration time (CMT) with coefficients of variation (RSD %; n = 5) of <1% for most of the proteinaceous compounds analyzed and showed greater effectiveness in discriminating paprika varieties than the SDS-PAGE technique. Four peaks found in the FZCE electropherograms were investigated as a basis for detecting and estimating the adulteration of smoked paprika with paprika elaborated from the Papri Queen variety. The adulteration detection limits varied from 5 to 40% of the Papri Queen variety within a satisfactory working range of mixture (5-80%) sufficiently large to cover the adulteration levels of interest. The use of peak 6 as a marker for determining adulteration gave the best results, with an adulteration detection limit of 5-10% (w/w).     

Rapid Identification of HMW Glutenin Subunits from Different Hexaploid Wheat Species by Acidic Capillary Electrophoresis

High molecular weight glutenin subunits (HMW‐GS) from three hexaploid wheat species (AABBDD, 2n=6x=42, Triticum aestivum L., T. spelta L., and T. compactum L.) were separated and identified by acidic capillary electrophoresis (A‐CE) with phosphate‐glycine buffer (pH 2.5) in uncoated fused‐silica capillaries (50 μm, i.d. × 25.5 cm) at 12.5 kV and 40°C. The rapid separations (<15 min) of HMW‐GS with good repeatability (RSD < 2%) were obtained using a fast capillary rising protocol. All 17 HMW‐GS analyzed could be well separated and their relative migration orders were ranked. In particular, the good quality subunit pair 5+10 could be differentiated from poor quality subunit pair 2+12. In addition, the other three allelic pairs of 13+16, 17+18, and 7+8 subunits that were considered to have positive effects on dough properties, as well as three pairs of novel subunits 13+22*, 13*+19*, and 6.1+22.1 detected from spelt and club wheat, can also be readily separated and identified. An additional protein subunit presented in Chinese bread wheat cultivar Jing 411 and club wheat TRI 4445/75, respectively, was detected by both A‐CE and 2‐D gel electrophoresis (A‐PAGE × SDS‐PAGE), for which further identification is needed.     

Interaction Between Sorghum Protein Extraction and Precipitation Conditions on Yield,Purity, and Composition of Purified Protein Fractions

Sorghum proteins have the potential to be used as a bio‐industrial renewable resource for applications such as biodegradable films and packaging. This project was designed to evaluate the effect of interactions between sorghum protein extraction and precipitation conditions on the yield, purity, and composition of sorghum protein fractions. Proteins were extracted with 70% ethanol under nonreducing conditions, with ultrasound, or under reducing conditions using either sodium metabisulfite or glutathione as the reducing agent. Several conditions were used to isolate the extracted proteins through precipitation, including lowering ethanol concentrations alone or in combination with lowering to pH 2.5, or by adding 1M NaCl to the extract. Combinations of these conditions were also tested. All precipitation conditions effectively precipitated proteins and lowering the pH and adding 1M NaCl to the extracts enhanced precipitation in some cases. However, the conditions that precipitated the maxium amount of protein or highest purity of protein varied according to how the proteins were initially extracted. Precipitated proteins were characterized by RP‐HPLC, SEC, HPCE, and SDS‐PAGE to compare the protein fractions composition. Nonreduced and sonicated samples had a much wider M_w distribution than reduced extracts. Thus, extraction and precipitation conditions influenced the isolated proteins yield, purity, and composition. Because the extraction and purification processes influenced the composition, purity, and biochemical properties, it may be possible to prepare protein fractions with unique functionalities for specific end‐uses.     

Separation of Wheat Proteins by Two-Dimensional Reversed-Phase High-Performance Liquid Chromatography Plus Free Zone Capillary Electrophoresis

Gliadins and glutenins from four hard red winter wheat cultivars were separated by a novel two-dimensional (2D) technique. Protein extracts were separated by reversed-phase high performance liquid chromatography as the first dimension with each 30-sec interval collected separately. Those fractions were then separated by free-zone capillary electrophoresis (FZCE) for the second dimension. Data was combined into 2D surface contour plots similar to traditional gel electrophoresis 2D maps. For HPLC, C₈ and C₁₈ columns were used in the first dimension to separate gliadins and glutenins, respectively. Uncoated fused silica capillaries (27 cm × 25 μm, i.d.) were used for the 2D FZCE separations. Differences in the 2D maps of both gliadin and glutenin fractions were found between pairs of both closely related and sister lines that varied in quality.     

Characteristics and Protein Subunit Composition of Flour Mill Streams from Different Commercial Wheat Classes and Their Relationship to White Salted Noodle Quality

Proximate characteristics and protein compositions of selected commercial flour streams of three Australian and two U.S. wheats were investigated to evaluate their effects on the quality of white salted noodles. Wheat proteins of flour mill streams were fractionated into salt‐soluble proteins, sodium dodecyl sulfate (SDS)‐soluble proteins, and SDS‐insoluble proteins with a sequential extraction procedure. SDS‐soluble proteins treated by sonication were subsequently separated by nonreducing SDS polyacrylamide gel electrophoresis (SDS‐PAGE). There was a substantial amount of variation in distributions of protein content and protein composition between break and reduction mill streams. SDS‐insoluble proteins related strongly to differences in protein quantity and quality of flour mill streams. The soluble protein extracted by SDS buffer included smaller glutenin aggregates (SDS‐soluble glutenin) and monomeric proteins, mainly gliadin (α‐, β‐, γ‐, and ω‐types) and albumin and globulin. SDS‐soluble proteins of different flour mill streams had similar protein subunit composition but different proportions of the protein subunit groups. Noodle brightness (L) decreased and redness (a) increased with increased SDS‐insoluble protein and decreased monomeric gliadin. Noodle cooking loss and cooking weight gain decreased with increased glutenin aggregate (SDS‐soluble glutenin and SDS‐insoluble glutenin) and decreased monomeric gliadin. Noodle hardness, springiness, cohesiveness, gumminess, chewiness, tensile strength, breaking length, and area under the tensile strength versus breaking length curve increased with increased glutenin aggregate. Monomeric gliadin contributed differently to texture qualities of cooked noodles from glutenin aggregate. Monomeric albumin and globulin were not related to noodle color attributes (except redness), noodle cooking quality, and texture qualities of cooked noodles. The results suggested that variation in protein composition of flour mill streams was strongly associated with noodle qualities.     

Separation and Quantification of HMW Glutenin Subunits by Capillary Electrophoresis

The use of capillary electrophoresis in SDS (SDS‐CE) for separation and quantification of HMW glutenin subunits (HMW‐GS) was investigated. HMW‐GS were precipitated with 40% acetone from 50% 1‐propanol extract of flour under reducing conditions after removal of monomeric proteins with 50% 1‐propanol. Poly (ethylene oxide) was used in the running buffer (3% w/v) for SDS‐CE. The results indicated that HMW‐GS could be well separated by SDS‐CE, including subunits 7+8, 7+9, 2+12, 5+10, and 17+18. However, HMW‐GS showed delayed migration times compared with molecular weight protein standards. Some HMW‐GS were reversed in their mobilities in SDS‐CE compared with their mobility and molecular weights by SDS‐PAGE. Therefore, the SDS‐CE was unsuitable for MW determination of HMW‐GS. A linear response was obtained from SDS‐CE of a plot of the concentration of HMW‐GS of the 40% acetone precipitate versus corrected areas for absorbance at 214 nm. Quantification of HMW‐GS for the two biotypes (subunits 5+10 vs. 2+12) of an Australian wheat cultivar Warigal confirmed the differences between the two biotypes in their quantity of HMW‐GS. Therefore, the technique could be used to quantify HMW‐GS in conjunction with SDS‐PAGE.     

Storage of Wheat Grains at Elevated Temperatures Increases Solubilization of Glutenin Subunits

Grains of two wheat (Triticum aestivum L.) cultivars, Sunco and Sunsoft, were stored at 4°C and 30°C for 270 days to examine changes in proteins during storage. When whole meal flour extracted from the grains was analyzed using an unfractionated protein extraction procedure, no significant changes were found in protein content or SDS‐PAGE profile for either cultivar in samples stored at 30°C compared with those stored at 4°C. Fractionation of the flour samples from stored grain into soluble and insoluble proteins revealed increases in soluble protein content for both cultivars stored at 30°C compared with 4°C. The soluble protein content, expressed as a percentage of the total protein, increased by 1.5% (P = 0.032) for Sunco and by 8.0 % (P = 0.158) for Sunsoft during storage at 30°C compared with those samples stored at 4°C. Analysis by SDS‐PAGE and subsequent protein identification revealed that the most evident change that occurred during storage at 30°C was an increase in the content of high molecular weight glutenin subunits (HMW‐GS) in the soluble fraction. The potential effect of changes in solubility of HMW‐GS on functional properties is discussed.     

Separation of olive proteins combining a simple extraction method and a selective capillary electrophoresis (CE) approach: application to raw and table olive samples

A simple extraction method was developed to extract proteins from olive samples based on chloroform/methanol extraction followed by a protein precipitation with cold acetone. Then, a capillary electrophoresis (CE) method was carried out using an acid buffer (1 M formic acid at pH 2) to ensure a positive net charge for proteins and a neutral charge for potential interferents as polyphenols. The method developed was applied to raw and table olive samples. Interestingly, raw olive samples showed differences in protein profiles depending upon the botanical variety of olives and their geographical region. Protein profiles obtained for table olives also showed differences according to the sample treatment. Thus, a signal reduction in the electropherograms obtained for black olives was observed in comparison to those achieved for treated green olives. In this work, the use of protein profiles was demonstrated to be a powerful tool for studying variations among olive samples.     

Capillary Electrophoresis as a Tool for Evaluating Lactic Acid Production from Sorghum

Sorghum is an underutilized resource for the production of bioindustrial chemicals like lactic acid. Capillary electrophoresis (CE) was tested to monitor the fermentation process, i.e., quantify the amount of lactic acid and by‐products in the fermentation broth using phosphate buffer pH 6.25 containing the electroosmotic flow modifier cetyltrimethylammonium bromide (CTAB). High levels of calcium carbonate were required during fermentation to stabilize the pH and these caused considerably prolonged migration times in CE. A 1:10 dilution of the samples with water was the best way to reduce salt load and thus conductivity in the sample plug, and thus to eliminate the problem of prolonged migration times. Further improvements were achieved by rinsing the capillary with HCl and water after each run, rather than NaOH and water. HCl might more efficiently remove Ca²⁺ ions from the capillary surface. The fermentation broth studied was based on liquefied sorghum inoculated with Rhizopus oryzae. The main product was lactic acid (24.60 ± 0.56 g/L) and a significant by‐product was fumaric acid (1.07 ± 0.04 g/L).     

Wheat Flour Proteins as Affected by Transglutaminase and Glucose Oxidase

Enzymes are good tool to modify wheat proteins by creating new bonds between the protein chains. In this study, the effect of the addition of glucose oxidase (GO) and transglutaminase (TG) on the wheat flour proteins is presented. The modification of wheat proteins was determined by analyzing the changes in gluten quality, alveograph parameters, and protein modifications. The amount of wet gluten increased with the addition of GO and TG, but the gluten quality was not improved in any case. Regarding the alveograph parameters, the effect of GO was readily evident obtaining wheat dough with higher tenacity and lower extensibility than the control, while TG led to doughs with lower tenacity and that were also less extensible. The protein modifications were characterized by free‐zone capillary electrophoresis (FZCE). FZCE data indicated that TG polymerizes mainly glutenins and, of those, the high molecular weight glutenin subunits were the most affected.     

Soil organic phosphorus in tropical forests: an assessment of the NaOH–EDTA extraction procedure for quantitative analysis by solution 31P NMR spectroscopy

The extraction of soil organic phosphorus by the NaOH–EDTA procedure was assessed in detail for a tropical forest soil (clay‐loam, pH 4.3, total carbon 2.7%). Optimum conditions for the quantification of soil organic phosphorus and characterization of its composition by solution ³¹P NMR spectroscopy were extraction in a solution containing 0.25 m NaOH and 50 mm Na₂EDTA in a 1:20 solid to solution ratio for 4 hours at ambient laboratory temperature. Replicate analyses yielded a coefficient of variation of 3% for organic phosphorus as a proportion of the spectral area. There was no significant difference in total phosphorus extraction from fresh and air‐dried soil, although slightly more organic phosphorus and less paramagnetic ions were extracted from dried soil. The procedure was not improved by changing the concentration of NaOH or EDTA, extraction time, or solid to solution ratio. Pre‐extraction with HCl or Na₂EDTA did not increase subsequent organic phosphorus extraction in NaOH–EDTA or improve spectral resolution in solution ³¹P NMR spectroscopy. Post‐extraction treatment with Chelex resin did not improve spectral resolution, but removed small concentrations of phosphorus from the extracts. Increasing the pH of NaOH–EDTA extracts (up to 1.0 m NaOH) increased the concentration of phosphate monoesters, but decreased DNA to an undetectable level, indicating its hydrolysis in strong alkali. The standardized NaOH–EDTA extraction procedure is therefore recommended for the analysis of organic phosphorus in tropical forest soils.     

Determination of organic acids and phosphate in soil aqueous extracts by capillary zone electrophoresis

Abstract

Organic acids and phosphate were determined in soil aqueous extracts by capillary zone electrophoresis using indirect UV detection. The electrolyte system was 10 mM sodium benzoate with 0.5 mM tetradecyltrimetyl‐ammonium bromide as flow modifier at pH 4.0, pH 4.5, or pH 5.0. This methodology was adequate to determine organic acids and phosphate in soil samples, but the major inorganic anions interfered in the determination. In all the samples of soil extracts, the presence of phosphate was detected. Acetate was found in most of the samples and lactate and formate in some of them.     

Development of liquid chromatography mass spectrometry method for analysis of organic N monomers in soil

The aim of this study was to develop an analytical procedure based on liquid chromatography-mass spectrometry (LC–MS) for analysis of monomeric organic N compounds in soil extracts. To benchmark the developed LC–MS method it was compared with a capillary electrophoresis–mass spectrometry (CE–MS) method recently used for analysis of small organic N monomers in soil. The separation was optimized and analytical performance assessed with 69 purified standards, then the LC–MS method was used to analyse soil extracts. Sixty-two out of 69 standards were analysable by LC–MS with separation on a hydrophilic interaction liquid chromatography column. The seven compounds that could not be analysed were strongly cationic polyamines. Limits of detection for a 5 μL injection ranged between 0.002 and 0.38 μmol L⁻¹, with the majority (49 out of 62) having limits of detection better than 0.05 μmol L⁻¹. The overall profile and concentration of small organic N monomers in soil extracts was broadly similar between LC–MS and CE–MS, with the notable exception of four ureides that were detected by LC–MS only. In soil extracts that had been concentrated ten-fold the detection and quantification of (some) organic N compounds was compromised by the presence of large amounts of inorganic salts. The developed LC–MS method offered advantages and disadvantages relative to CE–MS, and a combination of the two methods would achieve the broadest possible coverage of organic N in soil extracts.