Species Differentiation and Quantification of Processed Animal Proteins and Blood Products in Fish Feed Using an 8-Plex Mass Spectrometry-Based Immunoassay

Abstract

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.

Application of Mass Spectrometry-Based Immunoassays for the Species- and Tissue-Specific Quantification of Banned Processed Animal Proteins in Feeds

“…This set of ruminant-specific peptides offers the possibility for an unambiguous tissue detection using a 7-plex assay. Moreover, a second 8-plex assay for eight A2M-specific peptides was used here in this study to identify the species of the proficiency test samples . All peptide sequences and m / z ratios of precursor and fragment ions used in this study are given in Tables S2 and S3.…”

“…The bioinformatical selection of the ruminant-specific targets SERPINF2, C9, HP252, and A2M was reported earlier . The selection of homologous A2M-peptides of different species and the generation of a cross-species antibody we reported recently . In this study, we performed an untargeted shotgun analysis of bovine citrate plasma, milk, and MBM, respectively.…”

“…These targets were considered to increase the tissue specificity. In combination with an 8-plex assay for species differentiation, 56 we developed a two-tier process, which can detect species-specific PAPs and the tissue origin of bovine material. We analyzed six official ring trial samples for the presence of PAPs and its tissue origin with the novel analytical two-tier workflow, and concentrations down to a level of 0.1% PAP in feed (w/w) were detectable.…”

Official Feed Control Linked to the Detection of Animal Byproducts: Past, Present, and Future

“…In the last 2 years, the development of mass-spectrometry-based methods applied to PAPs identification has benefited from increased interest. Investigations were conducted for the development of targeted methods based on the detection of peptide biomarkers − or untargeted approaches using direct spectral library comparisons . Generally, the 0.1% (w/w) level of detection was reached for the targeted MS approaches.…”

Matrix and Sampling Effects on Quantification of Protein Biomarkers of Drug-Induced Liver Injury

“…All samples were prepared in a 96well microtiter plate for proteolytic digestion and subsequent immunoaffinity enrichment. A detailed protocol for the IA-LC−MS/MS workflow is described elsewhere 21 with the following modifications: Briefly, 15 μL of serum or plasma sample was mixed with 95 μL of digestion buffer comprising 100 mM triethanolamine and 0.5% (w/v) n-octylglucoside. Accordingly, 15 μL of bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with a concentration of 60 mg/mL was added to 95 μL of digestion buffer as a matrix for the calibration curve and blank samples.…”