NMI Natural and Medical Sciences Institute at the University of Tuebingen , Reutlingen 72770 , Germany.
SIGNATOPE GmbH , Reutlingen 72770 , Germany.
Anal Chem. 2019 Mar 19;91(6):3902-3911. doi: 10.1021/acs.analchem.8b04652. Epub 2019 Feb 27.
Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of nonruminant PAPs for use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, because the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here, we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides, and LC-MS/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of six ring trial samples obtained in former PCR and microscopy proficiency tests. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.
动物蛋白制品（PAPs）被认为是一种可持续的蛋白质来源，可以改善牲畜和水产养殖饲料的营养状况。然而，自疯牛病（BSE）危机以来，这些蛋白质的使用受到了严格的监管。2013 年，非反刍动物动物蛋白制品重新被引入水产养殖，这就需要开发替代分析方法来确定物种来源和组织来源（合法或非法）。目前的官方方法——光学显微镜和聚合酶链式反应——无法满足这些要求。此外，未来的方法还需要定量，因为计划用准确的阈值来取代即将实施的零容忍概念。在这里，我们开发了一种基于 7 重串联质谱的免疫测定法，能够以组织和物种特异性的方式定量检测饲料中 0.1%（w/w）的反刍动物动物蛋白制品。该工作流程包括在悬浮液中对动物蛋白制品进行 2 小时的胰蛋白酶消化、肽的免疫亲和富集以及基于 LC-MS/MS 的定量。结合之前发表的用于物种鉴定的测定法，我们能够确认六个来自以前的 PCR 和显微镜能力验证测试的环试验样品的物种和组织来源。所开发的测定法具有灵敏、定量、物种特异性和组织特异性，满足 PAP 检测新方法的要求，并可用于未来的饲料认证研究。
