Proteome profiling of L3 and L4 Anisakis simplex development stages by TMT-based quantitative proteomics

Abstract

Anisakis simplex is a parasitic nematode, which is a threat to the health of people all over the world [1]. The consumption of raw or inadequately prepared fish containing A. simplex larvae, due to their ability to penetrate the mucous membrane of the gastrointestinal trac t, as well as severe human allergic reactions, can cause illness called anisakiosis [2, 3]. 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: (a) extraction of the L3 and L4 larvae proteins, (b) trypsin digestion assisted with high intensity focused ultrasound (HIFU), (c) TMT-isobaric mass tag labeling, and (d) global proteome analysis (LC-MS/MS) of L3 and L4 A. simplex development stages using a LTQ-Orbitrap Elite mass spectrometer [4, 5, 6]. 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 were identified. Analysis of the most modulated proteins provided the specific proteomic signature of L3 (i.e. pseudocoelomic globin, endochitinase 1, paramyosin) and L4 (i.e. neprilysin-2, glutamate dehydrogenase, aminopeptidase N). To our knowledge, this is the most comprehensive dataset of proteins of A. simplex for two development stages (L3 and L4) identified to date. Obtained stage-specific proteins, could be used as targets to control/eliminate this parasite and in the future eradicate anisakiosis. Demonstrated (TMT) -based quantitative proteomics approach could be a universal tool to analyze global proteomic dynamics in parasitic nematode stages