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Mass Spectrometry Detection and Characterization of Partially Hydrolyzed Gluten
Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, a T-cell mediated immune disorder that affects about 1% of people worldwide. As therapeutic approaches are not currently available, a strict gluten-free diet is the only effective treatment for celiac patients. Consumers rely heavily on gluten-free labels to choose gluten-free food products, which need to contain less than 20 ppm gluten in the final products according to the FDA legislation. However, detection and quantification of gluten in fermented food products (i.e. malt, vinegar, sauces, beer, etc) can be very difficult since gluten has been partially hydrolyzed during food processing. The widely-used method for gluten detection, enzyme-linked immunosorbent assay (ELISA), may give false-negative results because the epitope(s) for gluten ELISA analysis might be hydrolyzed during fermentation. For a bottom-up MS analysis, the remaining gluten peptides may be too large for the identification. The complexity of gluten proteins, especially the large amount of highly homologous isoforms, presents an extra challenge for a top-down MS analysis. Therefore, an improved method is needed for the characterization and quantification of partially hydrolyzed gluten in fermented food products. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized, validated, and applied as a novel method for tracing gluten degradation throughout brewing and the in-depth characterization of partially hydrolyzed gluten in commercially available beers. Hydrolysis resistance peptides were consistently detected throughout the entire brewing processes and confirmed the concerns of wheat-based beers for celiac patients. Relatively large polypeptides in α/β gliadins and γ gliadins were predicted for investigated commercial beers and have provided information for developing a multiplexed ELISA that contains a panel of antibodies recognizing different portion of the proteins to determine the hydrolysis degree, potential celiac immunogenicity, and correct the gluten/gliadin or polypeptide concentration at different hydrolysis degrees simultaneously. This strategy also could be further optimized and applied to the characterization of partially hydrolyzed proteins in allergenic foods more broadly and may provide a reference for their safety assessment to both industry and regulatory authorities.
Cao, Wanying, "Mass Spectrometry Detection and Characterization of Partially Hydrolyzed Gluten" (2019). ETD collection for University of Nebraska - Lincoln. AAI27663590.