LC-MS/MS Reveals Hordeins Are Enriched in Brewers' Spent Grain.

Barley is commonly used in malting and brewing, and spent grain is repurposed for other foods. Barley contains gluten proteins called hordeins that cause intestinal damage and disease symptoms if eaten by people with celiac disease and related conditions. While the mashing process in brewing can partially hydrolyze immunogenic epitopes in hordeins, the immunogenic epitope load between the starting malt and spent grain has not been investigated. Herein, we quantified hordeins in commercially available spent grain and from matching malt. Liquid chromatography-mass spectrometry (LC-MS) and sandwich and competitive R5 ELISAs were used for quantification, revealing a higher abundance of gluten proteins in the spent grain product compared with the input malt. Certain hordein subtypes were enriched while others were depleted, and overall protein content was higher in spent grain. This suggests that the mashing process selectively extracts nonprotein components, leaving protein and hordein content elevated in spent grain. The spent grain products tested were not safe for consumers with celiac disease.

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.

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.

Hyperbaric oxygen as a model of lens aging in the bovine lens: The effects on lens biochemistry, physiology and optics.

Age related nuclear (ARN) cataracts in humans take years to form and so experimental models have been developed to mimic the process in animals as a means of better understanding the etiology of nuclear cataracts in humans. A major limitation with these animal models is that many of the biochemical and physiological changes are not typical of that seen in human ARN cataract. In this review, we highlight the work of Frank Giblin and colleagues who established an in vivo animal model that replicates many of the changes observed in human ARN cataract. This model involves exposing aged guinea pigs to hyperbaric oxygen (HBO), which by causing the depletion of the antioxidant glutathione (GSH) specifically in the lens nucleus, produces oxidative changes to nuclear proteins, nuclear light scattering and a myopic shift in lens power that mimics the change that often precedes cataract development in humans. However, this model involves multiple HBO treatments per week, with sometimes up to a total of 100 treatments, spanning up to eight months, which is both costly and time consuming. To address these issues, Giblin developed an in vitro model that used rabbit lenses exposed to HBO for several hours which was subsequently shown to replicate many of the changes observed in human ARN cataract. These experiments suggest that HBO treatment of in vitro animal lenses may serve as a more economical and efficient model to study the development of cataract. Inspired by these experiments, we investigated whether exposure of young bovine lenses to HBO for 15 h could also serve as a suitable acute model of ARN cataract. We found that while this model is able to exhibit some of the biochemical and physiological changes associated with ARN cataract, the decrease in lens power we observed was more characteristic of the hyperopic shift in refraction associated with ageing. Future work will investigate whether HBO treatment to age the bovine lens in combination with an oxidative stressor such as UV light will induce refractive changes more closely associated with human ARN cataract. This will be important as developing an animal model that replicates the changes to lens biochemistry, physiology and optics observed in human ARN cataracts is urgently required to facilitate the identification and testing of anti-cataract therapies that are effective in humans.

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.

Analysis of Gluten in Dried Yeast and Yeast-Containing Products.

Yeast are commonly used in the preparation of foods and beverages such as beer and bread and may also be used on their own as a source of nutrients and flavoring. Because of the historical connection of yeast to products made from wheat and barley, consumers maintaining a gluten-free diet can have concerns about the safety of yeast ingredients. Analyzing the safety of yeast and yeast-containing products presents some difficulties, as the yeast organisms actively degrade any gluten in the product, raising questions on the appropriateness of detection by traditional antibody-based methods. This study examines a variety of yeast and yeast-containing products by competitive ELISA and liquid chromatography-mass spectrometry for the estimated level of gluten proteins. While samples such as yeast extracts and nutritional yeast contained gluten levels below the 20 mg/kg (or parts per million, ppm) threshold defined by Codex Alimentarius, one baking yeast and a nutritional yeast supplement sample contained higher levels of gluten. This study demonstrates that both competitive ELISA and liquid chromatography-mass spectrometry provide similar results in the detection of wheat and barley gluten in yeast-containing products.

MALDI imaging mass spectrometry of ß- and γ-crystallins in the ocular lens.

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age-related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age-related modifications. Previous work demonstrated that, under standard protein IMS conditions, α-crystallin signals dominated the mass spectrum and age-related modifications to α-crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of ß- and γ-crystallins in ocular lens tissue. Acquired images showed that γ-crystallins were localized predominately in the lens nucleus whereas ß-crystallins were primarily localized to the lens cortex. Age-related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top-down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC-MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.

Spatial distributions of glutathione and its endogenous conjugates in normal bovine lens and a model of lens aging.

Glutathione (GSH) is the archetypal antioxidant, and plays a central role in the protection of the ocular lens from cataract formation. High levels of GSH are maintained in the transparent lens, but with advancing age, GSH levels fall in the lens nucleus relative to outer cortical cells, thereby exposing the nucleus of the lens to the damaging effects of oxygen radicals, which ultimately leads to age-related nuclear (ARN) cataract. Under normal conditions, GSH also forms endogenous conjugates to detoxify the lens of reactive cellular metabolites and to maintain cell homeostasis. Due to the intrinsic gradient of lens fibre cell age, the lens contains distinct regions with different metabolic requirements for GSH. To investigate the impact of fibre cell and lens aging on the varied roles that GSH plays in the lens, we have utilised high mass resolution MALDI mass spectrometry profiling and imaging analysis of lens tissue sections. High Dynamic Range (HDR)-MALDI FTICR mass spectrometry was used as an initial screening method to detect regional differences in lens metabolites from normal bovine lenses and in those subjected to hyperbaric oxygen as a model of lens aging. Subsequent MALDI imaging analysis was used to spatially map GSH and its endogenous conjugates throughout all lenses. Accurate mass measurement by MALDI FTICR analysis and LC-MS/MS mass spectrometry of lens region homogenates were subsequently used to identify endogenous GSH conjugates. While the distribution and relative abundance of GSH-related metabolic intermediates involved in detoxification pathways remained relatively unchanged upon HBO treatment, those involved in its antioxidant function were altered under conditions of oxidative stress. For example, reduced glutathione levels were decreased in the lens cortex while oxidised glutathione levels were elevated in the lens outer cortex upon HBO treatment. Interestingly, cysteineglutathione disulfide, was detected in the inner cortex of the normal lens, but was greatly decreased in the HBO-treated lenses. These results contribute to our understanding of the multiple roles that GSH plays in maintenance of lens transparency and in the age-related metabolic changes that lead to lens cataract formation.

Characterization of the Effects of Hyperbaric Oxygen on the Biochemical and Optical Properties of the Bovine Lens.

PURPOSE: To assess the morphologic, biochemical, and optical properties of bovine lenses treated with hyperbaric oxygen. METHODS: Lenses were exposed to hyperbaric nitrogen (HBN) or hyperbaric oxygen (HBO) for 5 or 15 hours, lens transparency was assessed using bright field microscopy and lens morphology was visualized using confocal microscopy. Lenses were dissected into the outer cortex, inner cortex, and core, and glutathione (GSH) and malondialdehyde (MDA) measured. Gel electrophoresis and Western blotting were used to detect high molecular weight aggregates (HMW) and glutathione mixed protein disulfides (PSSG). T2-weighted MRI was used to measure lens geometry and map the water/protein ratio to allow gradient refractive index (GRIN) profiles to be calculated. Optical modeling software calculated the change in lens optical power, and an anatomically correct model of the light pathway of the bovine eye was used to determine the effects of HBN and HBO on focal length and overall image quality. RESULTS: Lenses were transparent and lens morphology similar between HBN- and HBO-treated lenses. At 5- and 15-hour HBO exposure, GSH and GSSG were depleted and MDA increased in the core. Glutathione mixed protein disulfides were detected in the outer and inner cortex only with no appearance of HMW. Optical changes were detectable only with 15-hour HBO treatment with a decrease in the refractive index of the core, slightly reduced lens thickness, and an increase in optimal focal length, consistent with a hyperopic shift. CONCLUSIONS: This system may serve as a model to study changes that occur with advanced aging rather than nuclear cataract formation per se.