Validation of N-terminal Labeling Mass Spectrometry for Characterization of Partially Hydrolyzed Gluten Proteins

Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder which affects about 1% of people worldwide. The toxicity of partially hydrolyzed gluten (PHG) in fermented products such as beer is unknown due to the significant analytical challenges in PHG detection. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized for the in-depth analysis of PHG and validated using a test protease (trypsin) with known cleavage specificities. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated and native N-termini were confirmed by showing an MS1 peak area Log2 H:L with and without a significant difference (p < 0.01) from zero, respectively. Using the strategy, all abundant theoretical protease generated peptides in alpha/beta gliadins and gamma gliadins were identified. The validation indicated that the strategy can identify multiple protease cleavage sites in gluten and be used in subsequent analysis of yeast cleavage patterns in beer throughout brewing. This strategy can be further applied to characterize a broader partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.