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

Anisakis simplex is a parasitic nematode that can cause anisakiosis and/or allergic reactions in humans. The presence of invasive third-stage larvae (L3) in many different consumed fish species and the fourth-stage larvae (L4) in marine mammals, where L3 can accidentally affect to humans and develop as far as stage L4. World Health Organization and food safety authorities aim to control and prevent this emerging health problem. In the present work, using Tandem Mass Tag (TMT)-based quantitative proteomics we analyzed for the first time the global proteome of two A. simplex development stages, L3 and L4. The strategy was divided into four steps: (a) protein extraction of L3 and L4 development stages, (b) high intensity focused ultrasound (HIFU)-assisted trypsin digestion, (c) TMT-isobaric mass tag labeling following by high-pH reversed-phase fractionation, and (d) LC-MS/MS analysis in a LTQ-Orbitrap Elite mass spectrometer. A total of 2443 different proteins of A. simplex were identified. Analysis of the 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. SIGNIFICANCE: A. simplex is a fish-borne parasite responsible for the human anisakiosis and allergic reactions around the world. The work describes for the first-time the comparison of the proteome of two A. simplex stages (L3 and L4). The strategy is based on four steps: (i) protein extraction, (ii) ultra-fast trypsin digestion under High-Intensity Focused Ultrasound (HIFU), (iii) TMT-isobaric mass tag labeling followed by high-pH reversed-phase fractionation and (iv) peptide analysis using a LTQ-Orbitrap Elite mass spectrometer. The workflow allows to select the most modulated proteins as proteomic signature of those specific development stages (L3 and L4) of A. simplex. Obtained stage-specific proteins, could be used as targets to control/eliminate this parasite and in future eradicate the anisakiosis disease.