Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.
The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification.



To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient.
National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029, Nordnes, 5817 Bergen, Norway. Electronic address: josef.rasinger@nifes.no.
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JD
Rasinger
URBC-NARILIS, University of Namur, Namur, Belgium. Electronic address: helene.marbaix@unamur.be.
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Marbaix
URBC-NARILIS, University of Namur, Namur, Belgium; MaSUN, Mass spectrometry facility, University of Namur, Namur, Belgium. Electronic address: marc.dieu@unamur.be.
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Dieu
CRAW, Valorisation of Agricultural Products Department, 24 Chaussée de Namur, 5030 Gembloux, Belgium. Electronic address: o.fumiere@cra.wallonie.be.
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Fumière
CRAW, Biotechnology Department, 234 Chaussée de Charleroi, 5030 Gembloux, Belgium. Electronic address: se.mauro@cra.wallonie.be.
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Mauro
Center for Proteomics and Metabolomics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands. Electronic address: n.m.palmblad@lumc.nl.
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Palmblad
URBC-NARILIS, University of Namur, Namur, Belgium. Electronic address: martine.raes@unamur.be.
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Raes
National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029, Nordnes, 5817 Bergen, Norway. Electronic address: marc.berntssen@nifes.no.
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MHG
Berntssen
D000821:Animal Feed / Q000032:analysis* / Q000592:standards; D000818:Animals; D017756:Aquaculture / Q000379:methods; D002417:Cattle; D005398:Fisheries; D005399:Fishes; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D013045:Species Specificity
D011506:Proteins
Aquafeeds; Data mining; Feed safety; Processed animal protein; Shotgun proteomics