18: Mass spectrometry-based identification of allergen proteins involved in seafood related allergic reactions ​

Barletta, Elena1,2; Westermann​, Patrick1; Fröhlich, Klemens5,6; Brüggen, Marie-Charlotte3,4; Schmid-Grendelmeier, Peter​3,4; Bärenfaller, Katja1,2

  1. Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland
  2. Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland
  3. Christine Kühne-Center for Allergy Research and Education (CK-CARE), Davos, Switzerland
  4. Department of Dermatology, University Hospital, Zurich, Switzerland
  5. Institute of Surgical Pathology, Medical Center, University of Freiburg, Freiburg, Germany
  6. Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Shellfish are one of the most common causes of food allergies and a major cause of food-induced anaphylaxis. Approximately 2.5% of the world’s population has experienced an adverse reaction to seafood and the prevalence of seafood allergy is higher in populations residing in coastal geographic areas where seafood is an integral part of their diet. Sensitization and subsequent reactions occur most frequently upon food ingestion. However, they can also occur because of skin contact. Shrimps are, among all, the most consumed type of seafood worldwide and for that it is important to identify and characterize all possible allergens. Tropomyosin has been thought to be the most important allergen in shellfish, but also novel minor allergens have been discovered and characterized, and advances in component resolved diagnostics have provided insights into the prevalence of sensitization and their clinical importance in shellfish allergy.

As proteins consist of amino acid sequences, we will identify tropomyosin allergen proteins from peptides specific to seafood allergens. These peptide sequences can be only identified in seafood organism which share a high percentage of sequence identity and not in other organisms. As control we will also look for peptides from conserved protein regions. In our mass spectrometry-based experimental design, shrimp tissue is disrupted and homogenized, proteins are extracted, enzymatically digested, and subjected to Liquid Chromatography (LC) prior to Mass Spectrometry (MS). In this bottom-up proteomics approach the experimental mass spectra are then matched to peptides for protein identification, and these proteins are validated using a targeted MS-based proteomics approach called Parallel Reaction Monitoring (PRM), which is used to acquire high-resolution full MS/MS spectra for each target allergen peptide. In PRM a target precursor peptide is isolated in a quadrupole mass analyzer, fragmented in a collision cell and fragments are detected in an Orbitrap. Data analysis it then performed using conventional tools and as result we will obtain a chromatogram at each retention time point which consist of different transitions for all fragment ions, of one peptide precursor, displayed as colored lines identified in the legend.

Total protein extracts from shrimp (Penaeus monodon and Penaeus vannamei) are isolated and processed through in-gel tryptic digestion of SDS-PAGE gel fractions or using PreOmics columns with or without fractionation. Resulting peptides are then collected and purified prior to LC-MS/MS analysis and the MS raw files are processed by the SEQUEST algorithm within the protein database for decapods (TaxID = 6683). Tropomyosin proteins specific for shrimp, prawns, lobster, and crab are identified in our discovery workflow sharing a sequence identity between 89% and 100%. To support our findings, the PRM analysis is then performed looking for all shrimp unique tropomyosin peptides. A transition list for each peptide, from in silico digestion, is generated and analyzed within the Skyline open-source software. The same is performed for all other novel minor allergens identified within our MS analysis.

In all shrimp samples we accurately identified our proteins of interest. We established a MS-based proteomics workflow for the identification of proteins involved in seafood related allergic reactions. We were able to detect peptides from conserved protein region, e.g., shrimp actin (P60706), as well as peptides from protein regions specific to seafood allergens, e.g., shrimp tropomyosin (Q3Y8M6). It was possible to confirm the presence of the tropomyosin allergen as well as other minor allergens (arginine kinase, glyceraldehyde-3-phosphate dehydrogenase, sarcoplasmic Ca2+-binding protein, hemocyanin, troponin, etc.) with high confidence. The results obtained suggest the reproducibility of this proteomics workflow, so as to be used not only in the identification of other important allergens in seafood related allergic reactions, but also of allergens involved in other types of allergic diseases.