{"id":{"repo_id":"tu-berlin","oai_identifier":"oai:depositonce.tu-berlin.de:11303/25607"},"canonical_url":"https://search.dev.ndltd.org/etd/tu-berlin/oai:depositonce.tu-berlin.de:11303/25607","repository":{"repo_id":"tu-berlin","name":"Technische Universität Berlin","base_url":"https://api-depositonce.tu-berlin.de/server/oai/request"},"display":{"title":"Outlining different perspectives for accurate MS-based proteomics in processed matrices - a case study on European feed control","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Stobernack, Tobias"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Rappsilber, Juri"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025","date_published":"2025","updated_at":"2026-07-27T21:28:35Z","subjects":[],"languages":["en"],"rights":[],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://doi.org/10.14279/depositonce-24430"],"render_values":[{"text":"https://doi.org/10.14279/depositonce-24430","href":"https://doi.org/10.14279/depositonce-24430","code":true}]}]},"links":{"outbound_url":"https://depositonce.tu-berlin.de/handle/11303/25607","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Rappsilber, Juri"]},{"key":"dc:creator","label":"Author","values":["Stobernack, Tobias"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-09-16T14:20:59Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-09-16T14:20:59Z"]},{"key":"dc:date.issued","label":"Date","values":["2025"]},{"key":"dc:type","label":"Dc Type","values":["Doctoral Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://depositonce.tu-berlin.de/handle/11303/25607","https://doi.org/10.14279/depositonce-24430"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.","In Reaktion auf den Bovinen Spongiformen Enzephalopathie (BSE)-Ausbruchs in den 1990er Jahren schränkte die europäische Gesetzgebung den Einsatz tierischer Proteine als Futtermittel (FM) ein. Gesetzliche Kontrollmethoden können Regelverstöße nur begrenzt erfassen. Die Massenspektrometrie (MS) hat Potenzial zur Überwachung der Lebensmittel (LM)- und FM-Sicherheit, wobei ihre Sensitivität durch thermische Verarbeitung eingeschränkt sein kann. Diese Arbeit untersucht Strategien zur Verbesserung MS-gestützter Analysen in thermisch verarbeiteten Matrizes im Rahmen von bestehenden Lücken in der europäischen FM-Kontrolle. Ziel war zu prüfen, ob i) die Kombination von MS mit Magnetpartikel-basierter Immunoaffinitäts-Anreicherung (IAE) die Sensitivität in prozessierten Matrizes erhöht, ii) prozessierungs-induzierte Proteinmodifikationen in Rinder-LM- und FM-Zutaten zu identifizieren und deren Einfluss auf die MS-Quantifizierung abzuschätzen sowie iii) Verarbeitungsgrade blutbasierter FM anhand von Modifikationen zu differenzieren. Es wurde eine qualitativen MS-Methode zum Nachweis von Seidenraupenprotein in FM entwickelt. Drei spezies-spezifische Peptide wurden mittels Bottom-up-Proteomik identifiziert, Antikörper generiert und eine zielgerichtete Methode mit optionalem IAE-Schritt etabliert. Die Validierung ergab eine Nachweisgrenze (LOD) von ≤ 0,05% (m/m) in FM für Aquakultur, Geflügel und Schwein. IAE verbesserte das Signal-Rausch-Verhältnis bei niedriger Konzentration. Die Methode zeigte hohe Spezifität (auch bei Anwesenheit von zehn weiteren Insektenarten) sowie gute Reproduzierbarkeit (intra-/intertag Varianz ≤ 23%/38%). Die thermische Verarbeitung von FM und LM erschwert die MS-basierte Proteinquantifizierung und kann aufgrund proteinverändernder Reaktionen (z.B. Maillard-Reaktion, (Lipid)- Oxidation) zu erheblicher Unterbestimmung führen. Daher wurde der Einfluss der Prozessierung auf die MS-basierte Proteinquantifizierung untersucht. 37 bovine FMMaterialien (Fleisch, Knochen, Blut, Milch) in verschiedenen Verarbeitungsstufen (roh, sprühgetrocknet, drucksterilisiert) wurden hinsichtlich Proteinmodifikationen analysiert. Mehr als 300 Modifikationen (z.B. Oxidation, Deamidierung, Carboxymethylierung) wurden mittels klassischer (n = 53) und fehlertoleranter (n = 282) Datenbanksuche identifiziert. Der Einbezug modifizierter Varianten nativer Markerpeptide kann die Quantifizierungsgenauigkeit in verarbeiteten Matrizes erhöhen. Die europäische Gesetzgebung unterscheidet zwischen sprühgetrockneten Blutprodukten (BPs) und zum Teil drucksterilisierten (133 °C, 20 min, 3 bar) Blutmehlen (BMs). In einer Machbarkeitsstudie wurden in Schweine und Rinder BPs (n = 11) und BMs (n = 35) 53 verarbeitungsbedingte PTMs identifiziert. Nach der Auswahl anhand von Parametern und Normalisierung wurden fünf Schweine- und vier Rindermarker zur Unterscheidung von BPs und BMs identifiziert und deren Identität mit synthetischen Standards bestätigt. Die Ergebnisse dieser Arbeit verdeutlichen, dass MS-basierte Proteomik eine vielversprechende Methode zur Schließung bisheriger analytischer Lücken in der europäischen Futtermittelkontrolle darstellt."]},{"key":"dc:title","label":"Title","values":["Outlining different perspectives for accurate MS-based proteomics in processed matrices - a case study on European feed control"]}]}],"canonical_facts":{"dc:contributor.advisor":["Rappsilber, Juri"],"dc:creator":["Stobernack, Tobias"],"dc:date.accessioned":["2025-09-16T14:20:59Z"],"dc:date.available":["2025-09-16T14:20:59Z"],"dc:date.issued":["2025"],"dc:description.abstract":["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.","In Reaktion auf den Bovinen Spongiformen Enzephalopathie (BSE)-Ausbruchs in den 1990er Jahren schränkte die europäische Gesetzgebung den Einsatz tierischer Proteine als Futtermittel (FM) ein. Gesetzliche Kontrollmethoden können Regelverstöße nur begrenzt erfassen. Die Massenspektrometrie (MS) hat Potenzial zur Überwachung der Lebensmittel (LM)- und FM-Sicherheit, wobei ihre Sensitivität durch thermische Verarbeitung eingeschränkt sein kann. Diese Arbeit untersucht Strategien zur Verbesserung MS-gestützter Analysen in thermisch verarbeiteten Matrizes im Rahmen von bestehenden Lücken in der europäischen FM-Kontrolle. Ziel war zu prüfen, ob i) die Kombination von MS mit Magnetpartikel-basierter Immunoaffinitäts-Anreicherung (IAE) die Sensitivität in prozessierten Matrizes erhöht, ii) prozessierungs-induzierte Proteinmodifikationen in Rinder-LM- und FM-Zutaten zu identifizieren und deren Einfluss auf die MS-Quantifizierung abzuschätzen sowie iii) Verarbeitungsgrade blutbasierter FM anhand von Modifikationen zu differenzieren. Es wurde eine qualitativen MS-Methode zum Nachweis von Seidenraupenprotein in FM entwickelt. Drei spezies-spezifische Peptide wurden mittels Bottom-up-Proteomik identifiziert, Antikörper generiert und eine zielgerichtete Methode mit optionalem IAE-Schritt etabliert. Die Validierung ergab eine Nachweisgrenze (LOD) von ≤ 0,05% (m/m) in FM für Aquakultur, Geflügel und Schwein. IAE verbesserte das Signal-Rausch-Verhältnis bei niedriger Konzentration. Die Methode zeigte hohe Spezifität (auch bei Anwesenheit von zehn weiteren Insektenarten) sowie gute Reproduzierbarkeit (intra-/intertag Varianz ≤ 23%/38%). Die thermische Verarbeitung von FM und LM erschwert die MS-basierte Proteinquantifizierung und kann aufgrund proteinverändernder Reaktionen (z.B. Maillard-Reaktion, (Lipid)- Oxidation) zu erheblicher Unterbestimmung führen. Daher wurde der Einfluss der Prozessierung auf die MS-basierte Proteinquantifizierung untersucht. 37 bovine FMMaterialien (Fleisch, Knochen, Blut, Milch) in verschiedenen Verarbeitungsstufen (roh, sprühgetrocknet, drucksterilisiert) wurden hinsichtlich Proteinmodifikationen analysiert. Mehr als 300 Modifikationen (z.B. Oxidation, Deamidierung, Carboxymethylierung) wurden mittels klassischer (n = 53) und fehlertoleranter (n = 282) Datenbanksuche identifiziert. Der Einbezug modifizierter Varianten nativer Markerpeptide kann die Quantifizierungsgenauigkeit in verarbeiteten Matrizes erhöhen. Die europäische Gesetzgebung unterscheidet zwischen sprühgetrockneten Blutprodukten (BPs) und zum Teil drucksterilisierten (133 °C, 20 min, 3 bar) Blutmehlen (BMs). In einer Machbarkeitsstudie wurden in Schweine und Rinder BPs (n = 11) und BMs (n = 35) 53 verarbeitungsbedingte PTMs identifiziert. Nach der Auswahl anhand von Parametern und Normalisierung wurden fünf Schweine- und vier Rindermarker zur Unterscheidung von BPs und BMs identifiziert und deren Identität mit synthetischen Standards bestätigt. Die Ergebnisse dieser Arbeit verdeutlichen, dass MS-basierte Proteomik eine vielversprechende Methode zur Schließung bisheriger analytischer Lücken in der europäischen Futtermittelkontrolle darstellt."],"dc:identifier.uri":["https://depositonce.tu-berlin.de/handle/11303/25607","https://doi.org/10.14279/depositonce-24430"],"dc:language.iso":["en"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:title":["Outlining different perspectives for accurate MS-based proteomics in processed matrices - a case study on European feed control"],"dc:type":["Doctoral Thesis"]},"updated_at":"2026-07-27T21:28:35Z"}