Evaluation of N-terminal labeling mass spectrometry for characterization of partially hydrolyzed gluten proteins

Wanying Cao, Joseph L. Baumert, Melanie L. Downs

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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 is less well understood due to the significant analytical challenges in PHG characterization. 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 < .01) from zero and native N-termini with no significant difference from zero (p > .01). Using this strategy, all abundant, theoretical, test protease-generated peptides in exemplar alpha/beta gliadins and gamma gliadins were identified. Significance: This study is the first study that modified and evaluated TAILS analysis for the analysis of partially hydrolyzed gluten proteins. The evaluation indicated that the TAILS analysis could be modified and expanded to the identification of multiple protease cleavage sites in gluten proteins and is worth further evaluation as a novel strategy for the analysis of natural hydrolysis of gluten in food processes. 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.

Original languageEnglish (US)
Article number103538
JournalJournal of Proteomics
StatePublished - Jan 6 2020


  • Gluten
  • Hydrolyzed gluten
  • LC-MS/MS
  • Mass spectrometry
  • N-terminal labeling

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry


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