The epitopes in wheat proteins for defining toxic units relevant to human health.

Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The "epitope toxicity" value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

Signatures for torque variation in wheat dough structure are affected by enzymatic treatments and heating.

Molecular interactions in dough are poorly defined but affect final product usage. By monitoring changes in torque as dough is formed, we identified 80-85 °C as a gateway stage determining dough collapse during the mixing/heating process. We propose that this phenomenon is a diagnostic signature linked to integral features of dough complexes formed by some wheat varieties but not others. We found the dough at 80-85 °C was stabilized by increasing the starting bowl temperature (before a standard linear increase in temperature) of the mixing process and demonstrated the significance of specific macromolecular interactions that are formed early in the mixing process. Enzymes including papain, alpha-amylase, glucose oxidase and phytase stabilized dough structure to facilitate transition through the gateway temperatures between 80 and 85 °C. Our results show that if the dough initially formed a protein-starch complex that was too large, instability and collapse of the structure can occur later.

Preparation and Characterization of Avenin-Enriched Oat Protein by Chill Precipitation for Feeding Trials in Celiac Disease.

The safety of oats for people with celiac disease remains unresolved. While oats have attractive nutritional properties that can improve the quality and palatability of the restrictive, low fiber gluten-free diet, rigorous feeding studies to address their safety in celiac disease are needed. Assessing the oat prolamin proteins (avenins) in isolation and controlling for gluten contamination and other oat components such as fiber that can cause non-specific effects and symptoms is crucial. Further, the avenin should contain all reported immunogenic T cell epitopes, and be deliverable at a dose that enables biological responses to be correlated with clinical effects. To date, isolation of a purified food-grade avenin in sufficient quantities for feeding studies has not been feasible. Here, we report a new gluten isolation technique that enabled 2 kg of avenin to be extracted from 400 kg of wheat-free oats under rigorous gluten-free and food grade conditions. The extract consisted of 85% protein of which 96% of the protein was avenin. The concentration of starch (1.8% dry weight), ß-glucan (0.2% dry weight), and free sugars (1.8% dry weight) were all low in the final avenin preparation. Other sugars including oligosaccharides, small fructans, and other complex sugars were also low at 2.8% dry weight. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the proteins in these preparations showed they consisted only of oat proteins and were uncontaminated by gluten containing cereals including wheat, barley or rye. Proteomic analysis of the avenin enriched samples detected more avenin subtypes and fewer other proteins compared to samples obtained using other extraction procedures. The identified proteins represented five main groups, four containing known immune-stimulatory avenin peptides. All five groups were identified in the 50% (v/v) ethanol extract however the group harboring the epitope DQ2.5-ave-1b was less represented. The avenin-enriched protein fractions were quantitatively collected by reversed phase HPLC and analyzed by MALDI-TOF mass spectrometry. Three reverse phase HPLC peaks, representing ~40% of the protein content, were enriched in proteins containing DQ2.5-ave-1a epitope. The resultant high quality avenin will facilitate controlled and definitive feeding studies to establish the safety of oat consumption by people with celiac disease.

Characterization and comparative analysis of wheat high molecular weight glutenin subunits by SDS-PAGE, RP-HPLC, HPCE, and MALDI-TOF-MS.

High molecular weight glutenin subunits (HMW-GS) from 60 germplasms including 30 common wheat cultivars and 30 related species were separated and characterized by a suite of separation methods including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (RP-HPLC), high-performance capillary electrophoresis (HPCE), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Comparative analysis demonstrated that each methodology has its own advantages and disadvantages. The main drawback of SDS-PAGE was its overestimation of molecular mass and incorrect identification of HMW-GS due to its low resolution. However, it had the advantages of technical simplicity and low requirements of equipment; thus, it is suitable for large-scale and high-throughput HMW-GS screening for breeding programs, especially when the glutenin composition is clear in the breeding material. MALDI-TOF-MS clearly expressed many technical advantages among the four methods evaluated, including high throughput, high resolution, and accuracy; it was, however, associated with high equipment cost, thus preventing many breeding companies from accessing the technology. RP-HPLC and HPCE were found to be intermediate between SDS-PAGE and MALDI-TOF-MS. Both RP-HPLC and HPCE demonstrated higher resolution and reproducibility over SDS-PAGE but lower detection power than MALDI-TOF-MS. Results demonstrated that MALDI-TOF-MS is suitable for analyzing HMW-GS for routine breeding line screening and for identifying new genotypes.

Comparative field performance over 3 years and two sites of transgenic wheat lines expressing HMW subunit transgenes.

A series of transgenic wheat lines expressing additional high molecular weight (HMW) subunit genes and the corresponding control lines were grown in replicate field trials at two UK sites (Rothamsted Research, approximately 50 km north of London and Long Ashton, near Bristol) over 3 years (1998, 1999, 2000), with successive generations of the transgenic lines (T3, T4, T5) being planted. Four plots from each site were used to determine grain dry weight, grain nitrogen, dough strength (measured as peak resistance by Mixograph analysis) and the expression levels of the endogenous and "added" subunits. Detailed statistical analyses showed that the transgenic and non-transgenic lines did not differ in terms of stability of HMW subunit gene expression or in stability of grain nitrogen, dry weight or dough strength, either between the 3 years or between sites and plots. These results indicate that the transgenic and control lines can be regarded as substantially equivalent in terms of stability of gene expression between generations and environments.