Global proteome of L3 and L4 Anisakis simplex development stages: TMT-based quantitative proteomics. New approach in foodomics

Abstract

Anisakis simplex is a cosmopolitan parasitic nematode that can cause an illness called anisakiosis. A threat to the health of people all over the world may be the consumption of raw or inadequately prepared fish containing A. simplex larvae, due to their ability to penetrate the mucous membrane of the gastrointestinal tract, as well as severe human allergic reactions. The presence of invasive L3 larvae was documented in 200 species of fish and 25 species of cephalopods around the world, as well as the L4 stage in many species of marine mammals. New culinary trends involving the consumption of raw fish increase the geographical range of parasitic nematodes and the incidence of anisakiosis. Larvae are resistant to freezing, cooking, marinating and salting, which makes them difficult to eliminate. In this work, using TMT- based (tandem mass tags) quantitative proteomics the global proteome of L3 and L4 development stage of A. simplex was analyzed. The experiment was divided into four stages: (1) extraction of the L3 and L4 larvae proteins, (2) trypsin digestion assisted with high intensity focused ultrasound (HIFU), (3) TMT-isobaric mass tag labeling, and (4) global proteome analysis (LC-MS/MS) of L3 and L4 A. simplex development stages using a LTQ-Orbitrap Elite mass spectrometer. In this study, we create a reference proteome dataset for each of the two development stages of A. simplex, L3 and L4. Total of 2443 different proteins was identified, where the results showed a high degree of overlap (1542 different proteins) between L3 and L4 of A. simplex. In addition, a high amount of proteins specific only for L3 (330) or L4 (571) were identified and quantified. Gene ontology (GO) term was performed by the PANTHER classification system to understand the molecular function and biological processes of the identified proteins. Then, KEGG pathway analysis by DAVID 6.8 showed that most of the identified proteins in both stages: L3 and L4 were involved in main metabolic pathways (cel01100), ribosome (cel03010), biosynthesis of antibiotics (cel01130), carbon metabolism (cel01200) and oxidative phosphorylation (cel00190). The most complex nodes of the interaction network in the global proteome were those associated with energy metabolism, regulation of muscle contraction, protein catabolic processes, citrate cycle, aminoacyl-tRNA biosynthesis, and vesicle-mediated transport. The possible interactions were analyzed using the STRING v10.0 software. Due to the analysis of the specific proteins for L3 and L4 development stages of A. simplex, we identified and characterized many new proteins not yet assigned to this organism. These proteins participate in very important metabolic pathways for parasitic nematodes, which are essential for the development of the parasite. That makes them potential targets in research on antiparasitic substances, as well as may be used for the classification of food and feed contaminants. This valuable protein repository will add new and significant information to the universal public protein databases and will be very useful for further anisakiosis investigations, and eradication of A. simplex allergens from food, ensuring the safety of the consumers