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Corundum Particles as Trypsin Carrier for Efficient Protein Digestion

  • Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80 % across several reuse cycles, and storage at 4 °CReusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80 % across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved >60 % sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows.zeige mehrzeige weniger

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Metadaten
Autor*innen:Sarah DöringORCiD, Birte S. Wulfes, Aleksandra Atanasova, Carsten JaegerORCiD, Leopold Walzel, Georg TscheuschnerORCiD, Sabine Flemig, Kornelia GawlitzaORCiD, Ines FeldmannORCiD, Zoltán KonthurORCiD, Michael G. WellerORCiD
Dokumenttyp:Preprint
Veröffentlichungsform:Graue Literatur
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
1 Analytische Chemie; Referenzmaterialien / 1.9 Chemische und optische Sensorik
4 Material und Umwelt
4 Material und Umwelt / 4.2 Material-Mikrobiom Wechselwirkungen
Veröffentlichende Institution:Bundesanstalt für Materialforschung und -prüfung (BAM)
Verlag:Preprints.org
Erste Seite:1
Letzte Seite:22
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Technik, Medizin, angewandte Wissenschaften / Ingenieurwissenschaften / Ingenieurwissenschaften und zugeordnete Tätigkeiten
Freie Schlagwörter:Aluminum oxide; Antibodies; Enzyme immobilization; Mass spectrometry; Protein quantification
Themenfelder/Aktivitätsfelder der BAM:Chemie und Prozesstechnik
Chemie und Prozesstechnik / Chemische Charakterisierung und Spurenanalytik
Material
Material / Advanced Materials
DOI:10.20944/preprints202510.2002.v1
URN:urn:nbn:de:kobv:b43-647944
Verfügbarkeit des Dokuments:Datei für die Öffentlichkeit verfügbar ("Open Access")
Lizenz (Deutsch):License LogoCreative Commons - CC BY - Namensnennung 4.0 International
Datum der Freischaltung:24.11.2025
Referierte Publikation:Nein
Schriftenreihen ohne Nummerierung:Preprints der BAM
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