Scope: Gluten-containing grains cause adverse health effects in individuals with celiac disease. Fermentation of these grains results in gluten-derived polypeptides with largely uncharacterized sizes and sequences, which may still trigger an immune response. Materials and results: This research used N-terminal labeling mass spectrometry to identify protease cleavage sites during each stage of bench-scale brewing, including malting, mashing, boiling, fermentation, and aging. Gluten hydrolysates from each brewing step were tracked and the immunotoxic potential was evaluated in silico. The results indicate that proteolysis and precipitation of gliadins occurring during brewing differ by protein region and brewing stage. The termini of gliadins were hydrolyzed throughout the entire brewing process, but the central regions remained relatively stable. Most hydrolysis occurred during malting, and most precipitation occurred during boiling. The addition of yeast yielded new cleavage sites but did not result in complete hydrolysis. Consistent detection of peptides within the clinically important regions of gliadin corroborated the hydrolytic resistance of this region. Conclusions: N-terminal labeling mass spectrometry served as a novel approach to track the fate of gliadin/gluten throughout a bench-scale brewing process. Consistently identified fragments could serve as improved targets for detection of hydrolyzed gluten in fermented products.

Samples were collected before and after each of the following five steps of a bench-scale brewing process: malting, mashing, boiling, fermentation, and aging. Samples were prepared for analysis by conducting N-terminal labeling, molecular weight fractionation, secondary chymotryptic digestion and negative selection of labeled peptides. Briefly, samples collected before and after each brewing step were denatured, reduced, alkylated, and subjected to N-terminal dimethyl labeling with light or heavy formaldehyde, respectively. Light- and heavy-labeled samples were combined and fractionated with 30 kDa MWCO centrifugal filters. Proteins/polypeptides above 30 kDa were subsequently digested with chymotrypsin, and resulting unlabeled peptides were removed with an amine-binding polymer (HPG-ALD polymer). All samples underwent C18 clean-up before analysis by discovery mass spectrometry. Discovery proteomics was conducted using a top-10 data dependent acquisition method using a Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Peptide identification was conducting using the SEQUEST HT node in Proteome Discoverer 2.1.

(1) Wheat (soft white winter wheat, Brundage variety): before and after malting (2) Barley (two-row spring barley, Merit 57 variety): before and after malting (3) Mash suspension (wheat, barley): before and after mashing (4) Wort (wheat, barley, Hops): before and after boiling (5) Beer (wheat, barley, hops, yeast): before and after fermentation (6) Beer (wheat, barley, hops, yeast): before and after aging - All Samples Fractionated: Above and below 30 kDa