With the reintroduction of nonruminant processed animal proteins (PAPs) for use in aquaculture in 2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date, light microscopy and polymerase chain reaction are the official methods for proving the absence of illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species differentiation are needed. To address this issue, we developed and validated an 8-plex mass spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-macroglobulin peptides using a group-specific antibody, and a subsequent analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry for species identification and quantification. This workflow can be used to quantitatively determine the species origin in future feed authentication studies.

中文翻译：

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使用基于八重质谱的免疫分析法对鱼饲料中加工的动物蛋白和血液制品进行物种区分和定量

随着2013年非反刍动物加工动物蛋白（PAP）的重新引入以用于水产养殖，有一种合适的替代品来代替鱼饲料中的昂贵鱼粉。然而，由于牛海绵状脑病（BSE）危机，严格限制饲料中PAP的使用。迄今为止，光学显微镜和聚合酶链反应是证明饲料中不存在非法PAP的官方方法。由于它们的局限性，需要用于定量物种区分的替代方法。为了解决这个问题，我们开发并验证了一种基于8重质谱的免疫分析方法。工作流程包括对悬浮液中的PAP和血液制品进行胰蛋白酶消化，使用组特异性抗体对8种物种特异性α-2-巨球蛋白肽进行种间免疫亲和富集，随后通过超高效液相色谱与串联质谱联用进行分析，以进行物种鉴定和定量。此工作流程可用于在将来的饲料鉴定研究中定量确定物种起源。