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Reference proteomes of five wheat species as starting point for future design of cultivars with lower allergenic potential

Food Science and Technology

Reference proteomes of five wheat species as starting point for future design of cultivars with lower allergenic potential

M. Afzal, M. Sielaff, et al.

This groundbreaking study explored the flour proteome of five wheat species, revealing significant differences in protein expression linked to product quality and allergenic potential. Notably, einkorn wheat exhibited lower levels of allergens compared to common wheat. Conducted by researchers Muhammad Afzal, Malte Sielaff, Ute Distler, Detlef Schuppan, Stefan Tenzer, and C. Friedrich H. Longin, these findings can significantly aid in breeding healthier wheat cultivars.

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Playback language: English
Introduction
Wheat is a crucial staple food globally, contributing significantly to dietary protein intake. However, a substantial portion of the population experiences adverse reactions to wheat proteins, including celiac disease, wheat allergy, and non-celiac wheat sensitivity (NCWS). These reactions are often triggered by specific proteins such as gluten peptides, alpha-amylase/trypsin inhibitors (ATIs), serpins, lipid transfer proteins (LTPs), and beta-amylases. Previous research has focused on comparing specific protein families in modern and ancient wheat species or investigated the immunogenic potential of ATIs. This study aimed to comprehensively analyze the flour proteomes of five wheat species—common wheat, spelt, durum, emmer, and einkorn—using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differences in protein expression that could contribute to allergenicity and nutritional quality. The use of multiple cultivars grown in diverse environments aimed to capture both genetic and environmental influences on protein expression. This comprehensive analysis provides a robust foundation for the future development of wheat varieties with improved allergenic profiles and enhanced nutritional characteristics.
Literature Review
Existing literature includes studies comparing modern and ancient wheat species, often focusing on specific protein families like gluten proteins or ATIs. Some research highlighted differences in the immunogenic potential of ATIs between common wheat and einkorn. While earlier studies relied on gel-based proteomics, recent advancements in LC-MS-based proteomics allow for the quantification of thousands of proteins in a shorter timeframe. Recent proteomic studies have compared the proteomes of a limited number of cultivars within common wheat, spelt, and rye, revealing differences in protein expression even between closely related species. However, a comprehensive comparison of the flour proteomes of common wheat, spelt, durum, emmer, and einkorn using modern proteomic technology was lacking before this current study.
Methodology
The study utilized 150 flour samples from ten cultivars of each of five wheat species (common wheat, spelt, durum, emmer, and einkorn), each grown at three different locations in Germany/Austria. A label-free quantitative (LFQ) proteomics approach using high-resolution LC-MS/MS was employed. Protein extraction involved using a buffer containing urea, thiourea, CHAPS, and DTT. Samples were prepared using a filter-assisted sample preparation (FASP) protocol for tryptic digestion. LC-MS/MS analysis was performed using a nanoACQUITY UPLC system coupled to a SYNAPT G2-S mass spectrometer. Raw data were processed using ProteinLynx Global Server (PLGS) and ISOQuant for protein identification and quantification. Statistical analysis included linear mixed models to evaluate the effects of species, cultivars, and environments on protein expression, as well as t-tests to compare protein abundances between species. Hierarchical clustering was performed to group cultivars based on their proteomic profiles. Potential allergenic proteins were identified using information from databases such as the allergome database and a list of seed-borne wheat allergens, supplemented by the annotation of amylase/trypsin inhibitors (ATIs).
Key Findings
The study identified a total of 2,896 different proteins across all samples, representing a substantial proteome analysis in cereals. The number of proteins identified in each species was remarkably similar (approximately 2,540-2,700). However, hierarchical clustering clearly separated the five wheat species based on their proteome profiles, reflecting genetic distances. More than 50% of the proteins shared between any pair of species showed statistically significant differences in abundance. Einkorn wheat exhibited a significantly lower abundance of potential allergenic proteins (5.4-fold lower) and ATIs (7.2-fold lower) compared to common wheat. The abundance of these potential allergens was influenced more by genetics than by environment in many cases, indicating possibilities for breeding strategies. While environmental factors significantly impacted protein expression, a subset of proteins was found to have high heritability (>0.5), suggesting a strong genetic influence on their abundance and suitable for targeted breeding efforts. The analysis revealed that the allergenic protein content correlated closely with ploidy level, with diploid einkorn exhibiting the lowest levels.
Discussion
The findings highlight substantial proteome differences between wheat species, even among closely related ones. The significant reduction in potential allergens and ATIs in einkorn compared to common wheat warrants further investigation and suggests a potential for developing wheat varieties with lower allergenic potential. The significant influence of genetics on the expression of many proteins, particularly allergens, presents opportunities for targeted breeding programs. The detailed reference proteomes created in this study provide a valuable resource for future research on wheat allergy and breeding programs. However, the study's results need to be validated with clinical trials to confirm the improved tolerance of einkorn in individuals sensitive to wheat proteins. The environmental effect on protein abundance is crucial and underscores the need for considering diverse growing conditions when assessing protein expression.
Conclusion
This study provides a comprehensive reference proteome for five wheat species, revealing significant differences in protein expression that are potentially linked to allergenicity. Einkorn wheat stands out with its significantly lower levels of potential allergens and ATIs. The high heritability of many allergenic proteins opens possibilities for targeted breeding programs to reduce wheat allergenicity. Future research should focus on clinical trials to confirm the observed reductions in allergenic potential and explore the efficacy of einkorn in individuals with wheat sensitivities. Further research into the impact of flour processing methods on allergen levels is also warranted.
Limitations
The study used a limited number of technical replicates per sample due to budget and time constraints. While this was mitigated by the high number of biological replicates (10 cultivars per species across three environments), future studies could benefit from increasing the number of technical replicates. The lack of complete reference proteomes for some species might have resulted in an underestimation of unique proteins in those species. Further clinical trials are crucial to definitively confirm the health benefits and reduced allergenicity of einkorn wheat.
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