Celiac disease (CD) is one of the most frequent food hypersensitivities affecting approximately 1 % of the global population. CD is triggered by the ingestion of gluten from wheat, rye or barley. Currently, a lifelong gluten-free diet is the only possible therapy for this chronic inflammation of the small intestine. Intestinal tissue transglutaminase (TG2) plays a key role in the complex pathogenesis of CD. It forms, inter alia, covalently linked complexes with gluten peptides, which induce the formation of antibodies against these complexes. The binding sites of the gluten peptide-TG2 complexes consist of intermolecular Nε-(γ-glutamyl)-lysine bonds, so-called isopeptides. The aim of this study is to identify the isopeptides between TG2 and gluten peptides from wheat, rye and barley by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The characterization of gluten proteins and the development of a two-component model with human TG2 and gluten proteins laid the basis for the analysis of biologically formed isopeptides between gluten peptides and TG2. For this purpose, gastrointestinal digestion of the gluten proteins was simulated to obtain gluten peptides. To mimic the subsequent modifications of the gluten peptides by TG2, the gluten peptides were incubated with human TG2 in a second model simulating biological conditions. During this reaction, the gluten peptide-TG2 complexes were formed, which are linked via an isopeptide bond between a glutamine residue of the gluten peptide and the lysine residue of TG2. Subsequently, these complexes were hydrolyzed and the resulting peptides and isopeptides were analyzed by various LC-MS/MS methods. A total of 29 isopeptides were identified in 14 biological hydrolysates of gluten protein types of wheat, rye and barley incubated with TG2. The exact localization of the binding sites within the gluten peptides were already successfully determined by the non-targeted LC-MS/MS method for 18 isopeptides. These binding sites were reconfirmed by Parallel Reaction Monitoring (PRM) analysis and, in addition, nine more binding sites were accurately determined by this analytical technique. Some of the identified isopeptides contain known CD-specific sequences, which are related to the immune response of CD.

Identification of isopeptides between human tissue transglutaminase and gluten peptides

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