Peptidomics analysis of in vitro digested wheat breads: Effect of genotype and environment on protein digestibility and release of celiac disease and wheat allergy related epitopes.

Wheat proteins can trigger immunogenic reactions due to their resistance to digestion and immunostimulatory epitopes. Here, we investigated the peptidomic map of partially digested bread samples and the fingerprint of epitope diversity from 16 wheat genotypes grown in two environmental conditions. Flour protein content and composition were characterized; gastric and jejunal peptides were quantified using LC-MS/MS, and genotypes were classified into high or low bread protein digestibility. Differences in flour protein content and peptide composition distinguish high from low digestibility genotypes in both growing environments. No common peptide signature was found between high- and low-digestible genotypes; however, the celiac or allergen epitopes were noted not to be higher in low-digestible genotypes. Overall, this study established a peptidomic and epitope diversity map of digested wheat bread and provided new insights and correlations between weather conditions, genotypes, digestibility and wheat sensitivities such as celiac disease and wheat allergy.

Advancing Sustainable Malting Practices: Aquaporins as Potential Breeding Targets for Improved Water Uptake during Controlled Germination of Barley (Hordeum vulgare L.).

The conversion of raw barley (Hordeum vulgare L.) to malt requires a process of controlled germination, where the grain is submerged in water to raise the moisture content to >40%. The transmembrane proteins, aquaporins, influence water uptake during the initial stage of controlled germination, yet little is known of their involvement in malting. With the current focus on sustainability, understanding the mechanisms of water uptake and usage during the initial stages of malting has become vital in improving efficient malting practices. In this study, we used quantitative proteomics analysis of two malting barley genotypes demonstrating differing water-uptake phenotypes in the initial stages of malting. Our study quantified 19 transmembrane proteins from nine families, including seven distinct aquaporin isoforms, including the plasma intrinsic proteins (PIPs) PIP1;1, PIP2;1, and PIP2;4 and the tonoplast intrinsic proteins (TIPs) TIP1;1, TIP2;3, TIP3;1, and TIP3;2. Our findings suggest that the presence of TIP1;1, TIP3;1, and TIP3;2 in the mature barley grain proteome is essential for facilitating water uptake, influencing cell turgor and the formation of large central lytic vacuoles aiding storage reserve hydrolysis and endosperm modification efficiency. This study proposes that TIP3s mediate water uptake in malting barley grain, offering potential breeding targets for improving sustainable malting practices.

Utilizing proteomics to identify and optimize microalgae strains for high-quality dietary protein: a review.

Algae-derived protein has immense potential to provide high-quality protein foods for the expanding human population. To meet its potential, a broad range of scientific tools are required to identify optimal algal strains from the hundreds of thousands available and identify ideal growing conditions for strains that produce high-quality protein with functional benefits. A research pipeline that includes proteomics can provide a deeper interpretation of microalgal composition and biochemistry in the pursuit of these goals. To date, proteomic investigations have largely focused on pathways that involve lipid production in selected microalgae species. Herein, we report the current state of microalgal proteome measurement and discuss promising approaches for the development of protein-containing food products derived from algae.

Low Gluten Beers Contain Variable Gluten and Immunogenic Epitope Content.

Gluten content labels inform food choice and people practicing a gluten-free diet rely upon them to avoid illness. The regulations differ between jurisdictions, especially concerning fermented foodstuffs such as beer. Gluten abundance is typically measured using ELISAs, which have come into question when testing fermented or hydrolysed foodstuffs such as beer. Mass spectrometry can be used to directly identify gluten peptides and reveal false negatives recorded by ELISA. In this survey of gluten in control and gluten-free beers, gluten protein fragments that contain known immunogenic epitopes were detected using liquid chromatography-mass spectrometry in multiple beers that claim to be gluten-free and have sufficiently low gluten content, as measured by ELISA, to qualify as being gluten-free in some jurisdictions. In fact, several purportedly gluten-free beers showed equivalent or higher hordein content than some of the untreated, control beers. The shortcomings of ELISAs for beer gluten testing are summarised, the mismatch between ELISA and mass spectrometry results are explored, and the suitability of existing regulations as they pertain to the gluten content in fermented foods in different jurisdictions are discussed.

Proteome Changes Resulting from Malting in Hordein-Reduced Barley Lines.

Hordeum vulgare L., commonly known as barley, is primarily used for animal feed and malting. The major storage proteins in barley are hordeins, known triggers of celiac disease (CD). Here, sequential window acquisition of all theoretical mass spectra (SWATH)-MS proteomics was employed to investigate the proteome profile of grain and malt samples from the malting barley cultivar Sloop and single-, double-, and triple hordein-reduced lines bred in a Sloop background. Using a discovery proteomics approach, 2688 and 3034 proteins were detected from the grain and malt samples, respectively. By utilizing label-free relative quantitation through SWATH-MS, a total of 2654 proteins have been quantified from grain and malt. The comparative analyses between the barley grain and malt samples revealed that the C-hordein-reduced lines have a more significant impact on proteome level changes due to malting than B- and D-hordein-reduced lines. Upregulated proteins in C-hordein-reduced lines were primarily involved in the tricarboxylic acid cycle and fatty acid peroxidation processes to provide more energy for seed germination during malting. By applying proteomics approaches after malting in hordein-reduced barley lines, we uncovered additional changes in the proteome driven by the genetic background that were not apparent in the sound grain. Our findings offer valuable insights for barley breeders and maltsters seeking to understand and optimize the performance of gluten-free grains in malt products.

Conventional solid-state fermentation impacts the white lupin proteome reducing the abundance of allergenic peptides.

The demand for high-quality and sustainable protein sources is on the rise. Lupin is an emerging plant-based source of protein with health-enhancing properties; however, the allergenic potential of lupins limits their widespread adoption in food products. A combination of discovery and targeted quantitative proteome measurements was used to investigate the impact of solid-state fermentation induced by Rhizopus oligosporus on the proteome composition and allergenic protein abundances of white lupin seed. In total, 1,241 proteins were uniquely identified in the fermented sample. Moreover, the effectiveness of the solid-state fermentation in reducing the abundance of the tryptic peptides derived from white lupin allergens was demonstrated. Comparably, a greater decrease was noted for the major white lupin allergen based on ß-conglutin peptide abundances. Hence, conventional solid-state fermentation processing can be beneficial for reducing the potential allergenicity of lupin-based foods. This finding will open new avenues for unlocking the potential of this under-utilised legume.

Nutrition, allergenicity and physicochemical qualities of food-grade protein extracts from Nannochloropsis oculata.

Microalgae offer an opportunity to act as a sustainable source of dietary protein. This study aimed to evaluate the impact of different protein extraction methods on the nutritional and physicochemical properties of Nannochloropsis oculata. Food-grade protein extracts were obtained by hypotonic osmotic shock using milli-Q water. Food grade (FG) and non-food grade (NFG) extraction buffers were compared along with three cell disruption methods including bead beating, probe sonication and a combination of both methods for protein extraction. Mass spectrometry was used for protein and putative allergen identification in FG extracts. Bead beating led to a slightly higher number of identifiable proteins in FG extracts compared to control condition. Putative allergenic proteins were identified in FG extracts of N. oculata using different in-silico methods. These findings support the need to further evaluate the potential allergenic proteins in microalgae including N. oculata such as immunoglobulin E (IgE) binding tests.

Unpacking the Proteome and Metaproteome of the Black Soldier Fly Larvae: Efficacy and Complementarity of Multiple Protein Extraction Protocols.

The larvae of the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), have demonstrated the ability to efficiently bioconvert organic waste into a sustainable source of food and feed, but fundamental biology remains to be discovered to exploit their full biodegradative potential. Herein, LC-MS/MS was used to assess the efficiency of eight differing extraction protocols to build foundational knowledge regarding the proteome landscape of both the BSF larvae body and gut. Each protocol yielded complementary information to improve BSF proteome coverage. Protocol 8 (liquid nitrogen, defatting, and urea/thiourea/chaps) was better than all other protocols for the protein extraction from larvae gut samples, and the exclusion of defatting steps yielded the highest number of proteins for the larval body samples. Protocol-specific functional annotation using protein level information has shown that the selection of extraction buffer can affect protein detection and their associated functional classes within the measured BSF larval gut proteome. A targeted LC-MRM-MS experiment was performed on the selected enzyme subclasses to assess the influence of protocol composition using peptide abundance measurements. Metaproteome analysis of the BSF larvae gut has uncovered the prevalence of two bacterial phyla: actinobacteria and proteobacteria. We envisage that using complementary extraction protocols and investigating the proteome from the BSF body and gut separately will expand the fundamental knowledge of the BSF proteome and thereby provide translational opportunities for future research to enhance their efficiency for waste degradation and contribution to the circular economy.

Proteomic exploration reveals a metabolic rerouting due to low oxygen during controlled germination of malting barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) is used in malt production for brewing applications. Barley malting involves a process of controlled germination that modifies the grain by activating enzymes to solubilize starch and proteins for brewing. Initially, the grain is submerged in water to raise grain moisture, requiring large volumes of water. Achieving grain modification at reduced moisture levels can contribute to the sustainability of malting practices. This study combined proteomics, bioinformatics, and biochemical phenotypic analysis of two malting barley genotypes with observed differences in water uptake and modification efficiency. We sought to reveal the molecular mechanisms at play during controlled germination and explore the roles of protein groups at 24 h intervals across the first 72 h. Overall, 3,485 protein groups were identified with 793 significant differentially abundant (DAP) within and between genotypes, involved in various biological processes, including protein synthesis, carbohydrate metabolism, and hydrolysis. Functional integration into metabolic pathways, such as glycolysis, pyruvate, starch and sucrose metabolism, revealed a metabolic rerouting due to low oxygen enforced by submergence during controlled germination. This SWATH-MS study provides a comprehensive proteome reference, delivering new insights into the molecular mechanisms underlying the impacts of low oxygen during controlled germination. It is concluded that continued efficient modification of malting barley subjected to submergence is largely due to the capacity to reroute energy to maintain vital processes, particularly protein synthesis.

Comparison of protein extraction protocols and allergen mapping from black soldier fly Hermetia illucens.

Exploration of important insect proteins - including allergens - and proteomes can be limited by protein extraction buffer selection and the complexity of the proteome. Herein, LC-MS/MS-based proteomics experiments were used to assess the protein extraction efficiencies for a suite of extraction buffers and the effect of ingredient processing on proteome and allergen detection. Discovery proteomics revealed that SDS-based buffer yields the maximum number of protein groups from three types of BSF samples. Bioinformatic analysis revealed that buffer composition and ingredient processing could influence allergen detection. Upon applying multi-level filtering criteria, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. A targeted LC-MRM-MS assay was developed for the pan-allergen tropomyosin and used to assess the influence of buffer composition and ingredient processing using peptide abundance measurements. SIGNIFICANCE: We demonstrated that the selection of protein extraction buffer and the processing method could influence protein yield and cross-reactive allergen detection from processed and un-processed black soldier fly (BSF) samples. In total, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. An LC-MRM-MS assay was developed for tropomyosin, indicating the importance of buffer selection and processing conditions to reduce BSF samples' allergenicity.

Proteome Phenotypes Discriminate the Growing Location and Malting Traits in Field-Grown Barley.

Barley is one of the key cereal grains for malting and brewing industries. However, climate variability and unprecedented weather events can impact barley yield and end-product quality. The genetic background and environmental conditions are key factors in defining the barley proteome content and malting characteristics. Here, we measure the barley proteome and malting characteristics of three barley lines grown in Western Australia, differing in genetic background and growing location, by applying liquid chromatography-mass spectrometry (LC-MS). Using data-dependent acquisition LC-MS, 1571 proteins were detected with high confidence. Quantitative data acquired using sequential window acquisition of all theoretical (SWATH) MS on barley samples resulted in quantitation of 920 proteins. Multivariate analyses revealed that the barley lines' genetics and their growing locations are strongly correlated between proteins and desired traits such as the malt yield. Linking meteorological data with proteomic measurements revealed how high-temperature stress in northern regions affects seed temperature tolerance during malting, resulting in a higher malt yield. Our results show the impact of environmental conditions on the barley proteome and malt characteristics; these findings have the potential to expedite breeding programs and malt quality prediction.

Digestibility of wheat alpha-amylase/trypsin inhibitors using a caricain digestive supplement.

Wheat is a major source of nutrition, though in susceptible people it can elicit inappropriate immune responses. Wheat allergy and non-celiac wheat sensitivity are caused by various wheat proteins, including alpha-amylase trypsin inhibitors (ATIs). These proteins, like the gluten proteins which can cause celiac disease, are incompletely digested in the stomach such that immunogenic epitopes reach the lower digestive system where they elicit the undesirable immune response. The only completely effective treatment for these immune reactions is to eliminate the food trigger from the diet, though inadvertent or accidental consumption can still cause debilitating symptoms in susceptible people. One approach used is to prevent the causal proteins from provoking an immune reaction by enhancing their digestion using digestive protease supplements that act in the stomach or intestine, cleaving them to prevent or quench the harmful immune response. In this study, a digestive supplement enriched in caricain, an enzyme naturally present in papaya latex originally designed to act against gluten proteins was assessed for its ability to digest wheat ATIs. The digestion efficiency was quantitatively measured using liquid chromatography-mass spectrometry, including examination of the cleavage sites and the peptide products. The peptide products were measured across a digestion time course under conditions that mimic gastric digestion in vivo , involving the use of pepsin uniquely or in combination with the supplement to test for additive effects. The detection of diverse cleavage sites in the caricain supplement-treated samples suggests the presence of several proteolytic enzymes that act synergistically. Caricain showed rapid action in vitro against known immunogenic ATIs, indicating its utility for digestion of wheat ATIs in the upper digestive tract.

Evaluation of the Major Seed Storage Proteins, the Conglutins, Across Genetically Diverse Narrow-Leafed Lupin Varieties.

Lupin seeds have an excellent nutritional profile, including a high proportion of protein and dietary fiber. These qualities make lupin seeds an ideal candidate to help meet the growing global demand for complementary sources of protein. Of consequence to this application, there are nutritional and antinutritional properties assigned to the major lupin seed storage proteins-referred to as α-, ß-, δ- and γ-conglutins The variation in the abundance of these protein families can impact the nutritional and bioactive properties of different lupin varieties. Hence, exploring the conglutin protein profiles across a diverse range of lupin varieties will yield knowledge that can facilitate the selection of superior genotypes for food applications or lupin crop improvement. To support this knowledge generation, discovery proteomics was applied for the identification of the 16 known conglutin subfamilies from 46 domestic and wild narrow-leafed lupin (NLL) genotypes. Consequently, the diversity of abundance of these proteins was evaluated using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). This comparative study revealed a larger variability for the ß- and δ-conglutin content across the lines under study. The absence/lower abundance of the ß2- to ß6-conglutin subfamilies in a subset of the domesticated cultivars led to substantially lower overall levels of the allergenic ß-conglutin content in these NLLs, for which the elevation of the other conglutin families were observed. The diversity of the conglutin profiles revealed through this study-and the identification of potential hypoallergenic genotypes-will have great significance for lupin allergic consumers, food manufactures as well as grain breeders through the future development of lupin varieties with higher levels of desirable bioactive proteins and lower allergen content.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

Evaluation of protein extraction methods for in-depth proteome analysis of narrow-leafed lupin (Lupinus angustifolius) seeds.

Lupin is slated as a potential contributor towards future food security. Lupin possesses several nutritional and nutraceutical attributes, many linked to seed proteins. For in-depth characterisation of the lupin proteome, liquid chromatography-tandem mass spectrometry was used to evaluate four protein extraction procedures. The proteomes of three narrow-leafed lupin were qualitatively evaluated using protein/peptide identifications and further quantitatively assessed by data-independent proteome measurement. Each extraction buffer led to unique protein identifications; altogether yielding 2,760 protein identifications from lupin varieties. The analysis of protein abundance data highlighted distinct differences between Tris-HCl and urea extracted proteomes, while also revealing variation amongst the cultivar proteomes with the wild accession (P27255) distinctly different from the domesticated cultivars (Tanjil, Unicrop). The extraction buffer used influenced the proteome coverage, downstream functional annotation results and consequently the biological interpretation demonstrating the need to optimise and understand the impact of protein extraction conditions.

Immunoanalytic investigation of grain proteins antigenic for celiac disease patients in an einkorn collection.

Our study focuses on the complex characterization of a wild and cultivated einkorn collection of the Cereal Gene Bank of Agriculture Research Institute in Hungary, using proteomics, immune analytics and bioinformatics analyses. In a serological ELISA pre-screen of 208 different Triticum monococcum L. ssp. monococcum and Triticum monococcum L. ssp. aegilopoides genotypes with celiac disease samples high diversity was observed in the immune response. Based on the immune analytic results, four genotypes with significantly reduced immune reactivity were selected for detailed proteomics characterization. Our results confirm the benefits of high-throughput/large-scale pre-screening and the use of a complex examination platform to get relevant information about the genetic diversity of celiac disease-relevant proteins in the analyzed einkorn genotypes. These genotypes cannot be incorporated into the daily diet of celiac patients; however, they may represent candidates - especially in combination with enzymatic treatments - to improve the lifestyle of individuals suffering from other clinical conditions like non-celiac wheat sensitivity.

Yield-Related QTL Clusters and the Potential Candidate Genes in Two Wheat DH Populations.

In the present study, four large-scale field trials using two doubled haploid wheat populations were conducted in different environments for two years. Grain protein content (GPC) and 21 other yield-related traits were investigated. A total of 227 QTL were mapped on 18 chromosomes, which formed 35 QTL clusters. The potential candidate genes underlying the QTL clusters were suggested. Furthermore, adding to the significant correlations between yield and its related traits, correlation variations were clearly shown within the QTL clusters. The QTL clusters with consistently positive correlations were suggested to be directly utilized in wheat breeding, including 1B.2, 2A.2, 2B (4.9-16.5 Mb), 2B.3, 3B (68.9-214.5 Mb), 4A.2, 4B.2, 4D, 5A.1, 5A.2, 5B.1, and 5D. The QTL clusters with negative alignments between traits may also have potential value for yield or GPC improvement in specific environments, including 1A.1, 2B.1, 1B.3, 5A.3, 5B.2 (612.1-613.6 Mb), 7A.1, 7A.2, 7B.1, and 7B.2. One GPC QTL (5B.2: 671.3-672.9 Mb) contributed by cultivar Spitfire was positively associated with nitrogen use efficiency or grain protein yield and is highly recommended for breeding use. Another GPC QTL without negatively pleiotropic effects on 2A (50.0-56.3 Mb), 2D, 4D, and 6B is suggested for quality wheat breeding.

Proteome and Nutritional Shifts Observed in Hordein Double-Mutant Barley Lines.

Lysine is the most limiting essential amino acid in cereals, and efforts have been made over the decades to improve the nutritional quality of these grains by limiting storage protein accumulation and increasing lysine content, while maintaining desired agronomic traits. The single lys3 mutation in barley has been shown to significantly increase lysine content but also reduces grain size. Herein, the regulatory effect of the lys3 mutation that controls storage protein accumulation as well as a plethora of critically important processes in cereal seeds was investigated in double mutant barley lines. This was enabled through the generation of three hordein double-mutants by inter-crossing three single hordein mutants, that had all been backcrossed three times to the malting barley cultivar Sloop. Proteome abundance measurements were integrated with their phenotype measurements; proteins were mapped to chromosomal locations and to their corresponding functional classes. These models enabled the prediction of previously unknown points of crosstalk that connect the impact of lys3 mutations to other signalling pathways. In combination, these results provide an improved understanding of how the mutation at the lys3 locus remodels cellular functions and impact phenotype that can be used in selective breeding to generate favourable agronomic traits.

Perennial Ryegrass Contains Gluten-Like Proteins That Could Contaminate Cereal Crops.

Background: To ensure safe consumption of gluten-free products, there is a need to understand all sources of unintentional contamination with gluten in the food chain. In this study, ryegrass (Lolium perenne), a common weed infesting cereal crop, is analysed as a potential source of gluten-like peptide contamination. Materials and Methods: Ten ryegrass cultivars were analysed using shotgun proteomics for the presence of proteins from the prolamin superfamily. A relative quantitative assay was developed to detect ryegrass gluten-like peptides in comparison with those found in 10 common wheat cultivars. Results: A total of 19 protein accessions were found across 10 cultivars of ryegrass for the protein families of PF00234-Tryp_alpha_amyl, PF13016-Gliadin, and PF03157-Glutenin_HMW. Protein and peptide homology searches revealed that gliadin-like peptides were similar to avenin and gamma-gliadin peptides. A total of 20 peptides, characteristic of prolamin superfamily proteins, were selected for liquid chromatography mass spectrometry (LC-MS) with multiple reaction monitoring (MRM). Only two of the monitored peptides were detected with high abundance in wheat, and all others were detected in ryegrass. Glutenin and alpha-amylase/trypsin inhibitor peptides were reported for the first time in ryegrass and were noted to be conserved across the Poaceae family. Conclusion: A suite of gluten-like peptides were identified using proteomics that showed consistent abundance across ryegrass cultivars but were not detected in wheat cultivars. These peptides will be useful for differentiating wheat gluten contamination from ryegrass gluten contamination.

Proteome Analysis and Epitope Mapping in a Commercial Reduced-Gluten Wheat Product.

Gluten related disorders, such as coeliac disease, wheat allergy and baker's asthma are triggered by proteins present in food products made from wheat and related cereal species. The only treatment of these medical illnesses is a strict gluten-free diet; however, gluten-free products that are currently available in the market can have lower nutritional quality and are more expensive than traditional gluten containing cereal products. These constraints have led to the development of gluten-free or gluten-reduced ingredients. In this vein, a non-GMO wheat flour that purports to contain "65% less allergenic gluten" was recently brought to market. The present study aims to understand the alteration of the proteome profile of this wheat flour material. Liquid chromatography-mass spectrometry was used to investigate the proteome profile of the novel wheat flour, which was contrasted to a wheat flour control. Using both trypsin and chymotrypsin digests and a combined database search, 564 unique proteins were identified with 99% confidence. These proteins and the specific peptides used to identify them were mapped to the wheat genome to reveal the associated chromosomal regions in the novel wheat flour and the mixed wheat control. Of note, several ω- and γ-gliadins, and low-molecular weight glutenins mapping to the short arm of chromosome 1, as well as α-gliadins from the chromosome 6 short arm were absent or expressed at lower levels in the novel wheat variety. In contrast, the high-molecular weight glutenins and α-amylase/trypsin inhibitors were notably more abundant in this variety. A targeted quantitation experiment was developed using multiple reaction monitoring assays to quantify 359 tryptic and chymotryptic peptides from gluten and related allergenic proteins revealing a 33% decrease of gluten protein content in the novel wheat flour sample in comparison to mixed wheat control. However, additional mapping of known allergenic epitopes showed the presence of 53% higher allergenic peptides. Overall, the current study highlights the importance of proteomic analyses especially when complemented by sequence analysis and epitope mapping for monitoring immunostimulatory proteins.