Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Pathogenesis of celiac disease: Identification of isopeptides by LC-MS/MS (#72)

Barbara Lexhaller 1 , Christina Ludwig 2 , Peter Koehler 1 , Katharina Scherf 1
  1. Leibniz Institute for Food Systems Biology at the Technical University of Munich, Freising, Germany
  2. Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Freising, Germany

Celiac disease (CD) is one of the most frequent food intolerances affecting approximately 1 % of the population. CD is triggered by the ingestion of gluten from wheat, rye or barley. The intestinal tissue transglutaminase (TG2) plays a key role in the complex pathogenesis of CD, because it forms, inter alia, covalently linked complexes with gluten peptides, which induce the formation of antibodies against these complexes. These antibodies in turn contribute to an increased adaptive immune response. [1]

The aim of this study is to identify the binding sites between TG2 and gluten peptides.

The binding sites of the gluten peptide-TG2-complexes consist of intermolecular Nε(γ-glutamyl)lysine bonds, so-called isopeptides.[2] For the development of an analytical strategy to identify isopeptides, samples of the digested complexes and of a negative control were measured with a nano-LC-MS/MS system in triplicate and the data were analyzed by the two proteomic software tools MaxQuant and Skyline. Within MaxQuant the data were searched for peptides and isopeptides, comparing samples and negative controls. Subsequently, the MaxQuant output data and the raw data were analyzed with Skyline to visualize the differences. In the next step, the measured MS/MS-spectra were searched for the calculated masses of the product ions, which were then assigned to the peptide by the software.

These experiments on the identification of isopeptides were focused on the development of a suitable strategy, whereby an analysis method with a model system and an isopeptide standard were achieved. Furthermore, four isopeptides between a microbial transglutaminase and the model peptide could be identified, first by manual search in the data sets and secondly by combining the proteomics tools MaxQuant and Skyline.

  1. Schuppan D, Junker Y, Barisani D, Gastroenterology 2009; 137: 1912-1933
  2. Fleckenstein B, Qiao SW, Larsen MR et al., J. Biol. Chem. 2004; 279: 17607-17616