Surface Immobilization of Recombinant Trypsin onto Corundum Particles for Optimized Antibody Digestion

  • Therapeutic monoclonal antibodies are the fastest-growing class of biological agents and the development of reliable analytical methods for their quantification is becoming increasingly important. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) represents one of the leading technologies for antibody quantification. The serin protease trypsin has emerged as the gold standard enzyme for digesting intact protein into peptides for this approach. However, many protocols exist that often lead to different results depending on the digestion procedure. In particular, the type and amounts of detergents added during sample preparation for protein unfolding before digestion is known to create undesirable structure modifications of proteins (e.g. carbamylation) and significant bias in measurement results by inhibiting enzyme activity. The source and nature of the enzyme itself can also have an influence, whereby immobilized variants are increasingly being used to improveTherapeutic monoclonal antibodies are the fastest-growing class of biological agents and the development of reliable analytical methods for their quantification is becoming increasingly important. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) represents one of the leading technologies for antibody quantification. The serin protease trypsin has emerged as the gold standard enzyme for digesting intact protein into peptides for this approach. However, many protocols exist that often lead to different results depending on the digestion procedure. In particular, the type and amounts of detergents added during sample preparation for protein unfolding before digestion is known to create undesirable structure modifications of proteins (e.g. carbamylation) and significant bias in measurement results by inhibiting enzyme activity. The source and nature of the enzyme itself can also have an influence, whereby immobilized variants are increasingly being used to improve their stability, minimize self-digestion and enable a higher substrate-enzyme ratio. Here, we explored the potential of a novel thermostable trypsin and surface-functionalised corundum particles for enhanced antibody digestion. For this purpose, the inactive zymogen was recombinantly produced in E. coli, activated with enterokinase digestion and finally affinity purified by benzamidine purification. The enzyme activity was analogous to commercially available MS-grade trypsin product. In addition, produced trypsin showed increased thermal stability under typical digestion conditions compared to reference product. Furthermore, APTES-silanization followed by glutaraldehyde modification of corundum surface with an additional blocking step prevents nonspecific protein adsorption during the protein digestion procedure and the immobilized enzyme still showed substrate activity after covalent binding. Compared to wild-type and a non-immobilized enzyme, the surface-functionalized thermostable variant showed increased activity at elevated temperatures. Thus, this novel type of trypsin particles promises to not only improve antibody digestion at high temperatures, but also to prevent self-digestion and enable the separation of trypsin from target peptides in LC–MS analysis.zeige mehrzeige weniger

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Autor*innen:Sarah DöringORCiD
Koautor*innen:Zoltán KonthurORCiD, Michael G. WellerORCiD, Carsten JaegerORCiD, Birte S. Wulfes, Tue Anh Hoang
Dokumenttyp:Posterpräsentation
Veröffentlichungsform:Präsentation
Sprache:Englisch
Jahr der Erstveröffentlichung:2025
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik
1 Analytische Chemie; Referenzmaterialien / 1.8 Umweltanalytik
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Antibody Quantification; Corundum; Enzyme Immobilization; LC-MS/MS
Themenfelder/Aktivitätsfelder der BAM:Chemie und Prozesstechnik
Chemie und Prozesstechnik / Chemische Charakterisierung und Spurenanalytik
Veranstaltung:ANAKON 2025
Veranstaltungsort:Leipzig, Germany
Beginndatum der Veranstaltung:10.03.2025
Enddatum der Veranstaltung:13.03.2025
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:26.06.2025
Referierte Publikation:Nein
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