University of Warmia and Mazury, Faculty of Biology and Biotechnology, Department of Biochemistry, Olsztyn, Poland.
Spanish National Research Council (CSIC), Marine Research Institute (IIM), Department of Food Technology, Vigo, Pontevedra, Spain.
J Proteomics. 2019 Jun 15;201:1-11. doi: 10.1016/j.jprot.2019.04.006. Epub 2019 Apr 9.
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.
简单线虫是一种寄生线虫，可引起人类的异尖线虫病和/或过敏反应。在许多不同食用鱼类物种中存在侵袭性第三期幼虫（L3）和海洋哺乳动物中的第四期幼虫（L4），L3 幼虫可能意外影响人类并发育到 L4 期。世界卫生组织和食品安全当局旨在控制和预防这一新兴健康问题。在本工作中，我们首次使用串联质量标签（TMT）定量蛋白质组学分析了两种简单线虫发育阶段（L3 和 L4）的全蛋白质组。该策略分为四个步骤：（a）L3 和 L4 发育阶段的蛋白质提取，（b）高强度聚焦超声（HIFU）辅助胰蛋白酶消化，（c）TMT-等压质量标签标记后高 pH 反相分级分离，（d）在 LTQ-Orbitrap Elite 质谱仪中进行 LC-MS/MS 分析。共鉴定出简单线虫的 2443 种不同蛋白质。对调节蛋白的分析提供了 L3（即假体腔球蛋白、内几丁质酶 1、副肌球蛋白）和 L4（即 Neprilysin-2、谷氨酸脱氢酶、氨肽酶 N）的特定蛋白质组特征。据我们所知，这是迄今为止鉴定的两种发育阶段（L3 和 L4）的简单线虫最全面的蛋白质数据集。重要意义：简单线虫是一种鱼类寄生虫，可导致全球范围内的人类异尖线虫病和过敏反应。该工作首次描述了两种简单线虫阶段（L3 和 L4）的蛋白质组比较。该策略基于四个步骤：（i）蛋白质提取，（ii）高强度聚焦超声（HIFU）下超快胰蛋白酶消化，（iii）TMT-等压质量标签标记后高 pH 反相分级分离，（iv）使用 LTQ-Orbitrap Elite 质谱仪进行肽分析。该工作流程允许选择最受调节的蛋白质作为这些特定发育阶段（L3 和 L4）的简单线虫的蛋白质组学特征。获得的阶段特异性蛋白质可作为控制/消除这种寄生虫的靶标，并在未来消除异尖线虫病。
