Immunoaffinity-Based Mass Spectrometry for the Species Identification and Quantification of Processed Animal Proteins in Feed

Abstract

The present work introduced immunoaffinity-based mass spectrometry to feed analysis and improved the detection of banned processed animal proteins (PAPs) in animal feed. Current analytical methods show deficiencies in either sensitivity, species and tissue specificity or quantification ability. To address this issue, a peptide-centric workflow that comprises a more efficient sample preparation, an immunoaffinity enrichment of species- and tissue-specific peptides, and a LC-MS/MS analysis for identification and quantification using stable isotope labeled standard peptides, was established. The release of peptides from poorly soluble PAPs and blood products was improved by a direct digestion in suspension. Further time-consuming clean-up steps are not necessary since reagents and salts are removed during the immunoenrichment. The enrichment also allows a fast peptide separation using short gradients with a 10 min cycle time and therefore an increased sample throughput. The species differentiation of the 8 livestock species cattle, sheep/goat, pig, horse, turkey chicken, duck and goose, was addressed in a multispecies approach. Therefore, a cross-species polyclonal antibody was generated, which is able to enrich 8 homologous peptides from processed meat and bone meal, blood meal and spray-dried plasma, hence allowing a comprehensive analysis of common feed additives. A second multiplex assay was developed to differentiate ruminant tissues by targeting 7 peptides of meat, bone, cartilage, blood and milk proteins. This allows a differentiation of legal and illegal ruminant protein additives. The assays’ basic analytical parameters were validated. Both assays showed a detection limit in the picomolar concentration range allowing a qualitative detection over 4 to 5 orders of magnitude and a quantification over 3 to 4 orders of magnitude. Depending on the tissue type, 0.05%-0.75% PAP was specifically and quantitatively determined in an animal feed background. The multiplex assays were finally applied to official proficiency test samples from the European Reference Laboratory for Animal Proteins (EURL-AP, Gembloux, Belgium). The developed assays showed an unambiguous differentiation and quantification of species and tissues on a contamination level of 0.1% PAP in feed. As a final conclusion, immunoaffinity-based mass spectrometry was shown to overcome the current limitations in PAP detection and meets the requirements for future feed authentication methods.