Outlining different perspectives for accurate MS-based proteomics in processed matrices - a case study on European feed control

Since the outbreak of Bovine Spongiform Encephalopathy (BSE) in the 1990s, European legislation has restricted animal-derived protein in feed. However, current official control methods only partially ensure compliance. Mass spectrometry (MS) offers a promising approach to ensure food and feed safety, though thermal processing may compromise its sensitivity. Hence, this thesis explores strategies to enhance MS-based analysis in thermally processed matrices, addressing key challenges in European feed control. The main objectives were to: i) evaluate if combining MS with bead-based immunoaffinity enrichment (IAE) enhances sensitivity in processed matrices; ii) identify processing-induced protein modifications in bovine food and feed ingredients and estimate their impact on MS quantification; iii) differentiate processing states of blood-derived ingredients by exploiting processing-induced modifications. Part I of the thesis presents the development of a qualitative MS method for detecting silkworm protein in feed. Three silkworm-specific peptides were identified via bottom-up proteomics, antibodies produced and a targeted method with optional IAE developed. Method validation showed a limit of detection (LOD) ≤ 0.05% (w/w) in aquaculture, poultry, and pig feed, with IAE enhancing signal-to-noise at the lowest tested concentration. The method demonstrated high specificity (including in the presence of ten other insect species) and reproducibility (intra-/inter-day variation ≤ 23%/38%). Workflow robustness and performance support its application in official feed control and suggest potential for broader use in the food sector. Thermal processing of feed ingredients challenges MS-based protein quantification and can lead to significant underestimation due to protein-altering reactions (e.g., Maillard reaction, (lipid) oxidation). In Part II, 37 bovine materials (meat, bone, blood, milk) subjected to varying processing degrees (raw, spray-dried, pressure-sterilized) were analyzed for protein posttranslational modifications (PTMs). More than 300 covalent protein changes (e.g., oxidation, deamidation, carboxymethylation) were identified via classical (n = 53) and error-tolerant (n = 282) database searches. Analysis of ruminant-specific marker peptides and their modified variants indicate that protein quantification accuracy in thermally treated matrices may be improved by incorporating two modified variants in addition to the native marker. European legislation distinguishes between typically spray-dried blood products (BPs) and blood meals (BMs), which may require pressure sterilization (133 °C, 20 min, 3 bar) to reduce BSE risk. Part III of the thesis investigates MS-based differentiation of processing levels via protein modifications in a proof-of-principle study. Porcine and bovine BPs and BMs were analyzed, identifying 53 processing-associated PTMs. After multi-stage filtering and normalization, five porcine and four bovine marker candidates were found to distinguish BPs from BMs. Marker identities were verified using synthetic standards. Overall, this thesis underscores the potential of MS-based proteomics to advance official European feed control by closing existing analytical gaps.

Author dc:creator

• Stobernack, Tobias

Advisor dc:contributor.advisor

• Rappsilber, Juri

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Identifier URI

https://doi.org/10.14279/depositonce-24430

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/25607