Validation of N-terminal Labeling Mass Spectrometry for Characterization of Partially Hydrolyzed Gluten Proteins
Cao W, Baumert JL, Downs ML. Evaluation of N-terminal labeling mass spectrometry for characterization of partially hydrolyzed gluten proteins. Journal of proteomics. 2019 Oct 17:103538.
- Organism: wheat
- Instrument: Q Exactive Plus
LC-MS/MS; Hydrolyzed gluten; gluten; N-terminal Labeling; mass spectrometry; TAILS
- Lab head:
Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder that 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 specificity. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated neo N-termini were identified by exhibiting an MS1 Log2 H:L peak area ratio with a significant difference (p < 0.01) from zero and native N-termini with no significant difference from zero (p > 0.01). Using this strategy, all abundant, theoretical, protease-generated peptides in alpha/beta gliadins and gamma gliadins were identified. This study is the first study that modified and validated TAILS analysis for the analysis of partially hydrolyzed gluten proteins. The validation indicated that the strategy can identify multiple protease cleavage sites in gluten and may be a useful analytical technique in subsequent analysis of yeast cleavage patterns in beer throughout brewing. This strategy also may be further applied to characterize a broader range of partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.
In this study, TAILS was modified and validated for the analysis of partially hydrolyzed gluten proteins using a test enzyme with a known cleavage specificity (trypsin). The sample processing procedure was further optimized to reduce the number of potential interference peptides. The coverage of the modified N-terminal labeling MS analysis for gluten was investigated by comparing an in-silico prediction of proteolysis resulting peptides and native N-termini peptides with those identified in the experimental N-terminal analysis.
Monomeric gliadins were chosen as the exemplary gluten source for this study. Commercially available gliadin was investigated and used for a digestion evaluation in this study. For MS analysis, gliadin was isolated from a hard white whole wheat flour purchased from a local market according to a modified Osborne procedure.
Created on 4/30/19, 4:31 PM