LC-MS/MS quantitation of α-amylase/trypsin inhibitor CM3 and glutathione during wheat sourdough breadmaking.

AIMS: This study aimed to quantify α-amylase/trypsin inhibitor (ATI) CM3 and glutathione (GSH) during wheat sourdough breadmaking. METHODS AND RESULTS: Breads were made with two wheat cultivars and fermented with Fructilactobacillus sanfranciscensis, F. sanfranciscensis ΔgshR or Latilactobacillus sakei; chemically acidified and straight doughs served as controls. Samples were analysed after mixing, after proofing and after baking. GSH and CM3 were quantified by multi-reaction-monitoring-based methods on an LC-QTRAP mass spectrometer. Undigested ATI extracts were further examined by SDS-PAGE. CONCLUSIONS: GSH abundance was similar after mixing and after proofing but increased after baking (p < 0.001), regardless of fermentation. In breads baked with cv. Brennan, the samples fermented with lactobacilli had higher GSH abundance (p < 0.001) than in the controls. CM3 relative abundance remained similar after mixing and after proofing but decreased after baking (p < 0.001) across all treatments. This trend was supported by the SDS-PAGE analysis in which ATI band intensities decreased after baking (p < 0.001) in all experimental conditions. The overall effect of baking exerted a greater effect on the abundances of GSH and CM3 than fermentation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to quantify ATI over the course of breadmaking by LC-MS/MS in sourdough and straight dough processes.

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in replication and evaluated the reproducibility and precision of the workflow for the quantification of ATIs. Applying the method to analyze different wheat species (i.e., common wheat, spelt, durum wheat, emmer, and einkorn) and comparing the results to published data, we validated inter-laboratory and cross-methodology reproducibility of ATI quantification, which is essential in the context of large-scale breeding projects. Additionally, we applied our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of same varieties grown at different locations. Finally, we explored the potential of combining QconCAT-based absolute quantification with DIA-based LFQ proteome analysis for the generation of new hypotheses or assay development.

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.

Wheat ATI CM3, CM16 and 0.28 Allergens Produced in Pichia Pastoris Display a Different Eliciting Potential in Food Allergy to Wheat ‡.

Although wheat is a staple food for most of the human population, some of its components trigger adverse reactions. Among wheat components, the alpha-amylase/trypsin inhibitors (ATI) are important triggers of several allergies and activators of innate immunity. ATI are a group of exogenous protease inhibitors and include several polypeptides. The three ATI polypeptides named CM3, CM16 and 0.28 are considered major allergens, and might also play a role in other common wheat-related pathologies, such as Non Celiac Wheat Sensitivity and even Celiac Disease. On this basis, we pointed to obtain high amounts of them in purity and to evaluate their allergenicity potential. We thus isolated the mRNA corresponding to the three ATI genes CM3, CM16 and 0.28 from 28 days post-anthesis wheat kernels and the corresponding cDNAs were used for heterologous expression in Pichia pastoris. The three purified proteins were tested in degranulation assay against human sera of patients with food allergy to wheat. A large range of degranulation values was observed for each protein according to the sera tested. All of the three purified proteins CM3, CM16 and 0.28 were active as allergens because they were able to induce basophils degranulation on wheat allergic patients' sera, with the highest values of ß-hexosaminidase release observed for CM3 protein.

Tracking the fate of pasta (T. Durum semolina) immunogenic proteins by in vitro simulated digestion.

The aim of the present study was to identify and characterize the celiacogenic/immunogenic proteins and peptides released during digestion of pasta (Triticum durum semolina). Cooked pasta was digested using a harmonized in vitro static model of oral-gastro-duodenal digestion. The course of pasta protein digestion was monitored by SDS-PAGE, and gluten proteins were specifically analyzed by Western blot using sera of celiac patients. Among the allergens, nonspecific lipid-transfer protein was highly resistant to gastro-duodenal hydrolysis, while other digestion-stable allergens such as α-amylase/trypsin inhibitors were not detected being totally released in the pasta cooking water. To simulate the final stage of intestinal degradation, the gastro-duodenal digesta were incubated with porcine jejunal brush-border membrane hydrolases. Sixty-one peptides surviving the brush-border membrane peptidases were identified by liquid chromatography-mass spectrometry, including several gluten-derived sequences encrypting different motifs responsible for the induction of celiac disease. These results provide new insights into the persistence of wheat-derived peptides during digestion of cooked pasta samples.