For the millions of Americans afflicted with celiac disease, a diet devoid of gluten is the only successful treatment option. Analytical methods that are able to accurately quantitate hydrolyzed gluten in terms of intact gluten are needed to enforce compliance of the gluten-free labeling of fermented beverages. Additionally, proline endopeptidase (PEP) is an enzyme used by some brewers to purportedly degrade immunogenic gluten sequences in an attempt to produce beer that is safe for consumption by individuals with celiac disease. The goal of this study is to determine the effects of proline endopeptidase on gluten by using mass spectrometry to characterize the hydrolyzed gluten present in beers brewed with and without PEP. Sorghum beer incurred with 200 ppm wheat gluten was brewed with and without the addition of PEP at the start of fermentation. The beer samples were fractionated into the hydrolyzed peptides, which were analyzed directly by LC-MS/MS and database matching, and the intact proteins, which were analyzed using a traditional bottom-up proteomics approach via digestion with chymotrypsin. A number of gluten peptides were identified in both of the hydrolyzed peptide and intact protein fractions of the untreated and PEP treated samples, indicating that some gluten proteins remain, at least partially, intact after fermentation and enzymatic treatment. This observation confirms Western Blot results from these samples, which showed the presence of intact proteins after fermentation and enzymatic treatment. More hydrolyzed gluten peptides were identified in the PEP treated samples compared to the untreated samples, whereas more chymotryptic gluten peptides were identified in the untreated samples compared to the PEP treated samples. This suggests that PEP treatment does lead to increased degradation of intact gluten proteins; however the gluten was not degraded completely and gluten protein fragments and gluten peptides remained. The identified hydrolyzed and chymotryptic gluten peptides were compared to a database of known celiac disease relevant T-cell epitopes, using a custom python script, to determine how PEP treatment changed the profile of immunogenic gluten sequences in beer. A few known T-cell relevant epitopes were observed in the PEP treated samples, including an immunodominant peptide from the 33-mer.

Mass spectrometric analysis of the effects of a proline endopeptidase on gluten in a wheat gluten incurred model sorghum beer

254th National Meeting (2017)

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