Mass Spectrometry-Based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds

Abstract

The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR) are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of nonruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides, and a LC-MS/MS-based analysis for peptide quantification using isotope labeled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of 4 orders of magnitude with a detection limit in the range of 0.1% to 1% (w/w).

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

“…For a porcine blood meal and a porcine spray-dried plasma sample, the HPD protocol was compared with an extraction in the same buffer and subsequent in-solution digestion (ISD) of the supernatant. An absorption measurement at 280 nm indicated that HPD released more peptides compared to ISD, as was seen in the previous study for a bovine meat and bone meal sample . Peptide amounts released by HPD were nearly 2 times higher in BM and 5 times higher in SDP (data not shown).…”

“…PAP and blood product samples were prepared for LC-MS/MS analysis using a protocol we validated and published recently . Protein extraction and digestion were performed in a single step using heterogeneous phase digestion (HPD).…”

“…To further improve throughput and sensitivity, hybrid methods that combine immunoprecipitation with liquid chromatography coupled to mass spectrometry (LC-MS) have been established on both matrix-assisted laser desorption/ionization MS (MALDI-MS) and electrospray ionization MS (ESI-MS) platforms. , So far, immunoaffinity MS is used in clinical and pharmaceutical research for the quantification of receptors, kinases, drug-metabolizing enzymes, and plasma proteins . Recently, we introduced immunoaffinity MS to feed analysis for the species-specific quantification of banned ruminant PAPs in feed . Here, we apply this concept to enrich homologous peptides from different species in a cross-species epitope approach using only one antibody.…”

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra

“…Proteomic-based methods using (tandem) mass spectrometry (MS) were, in a recent scientific opinion by the European Food Safety Authority (EFSA), identified as promising tools to complement current standard techniques of feed PAP detection [18]. Different laboratories specialized in feed and food safety have been developing promising MS based tools for the species-specific detection, differentiation and quantification of animal proteins [19,20,21,22]. Most of these methods are targeted approaches which are based on the detection of a known peptide or protein of which the sequence information is available [23,24].…”

Identification and evaluation of potentially mutagenic and carcinogenic food contaminants

“…Research at the BfR in projects funded by the German Federal Ministry of Food and Agriculture currently also focuses on mass spectrometry methods for detection of terrestrial animal species in highly processed feed. Unlike the global spectral library‐based methods developed at the IMR, researchers at the BfR and its collaboration partner, the Natural and Medical Sciences Institute at the University of Tübingen (NMI), focus on targeted mass spectrometry analysis (Steinhilber et al., ). This approach allows a more accurate estimation of the abundance of selected PAP animal species in unknown samples, but strongly relies on the discovery of suitable marker peptides, which the IMR database can provide.…”