Zitieren Sie bitte immer diesen URN: urn:nbn:de:kobv:b43-478973
Efficient Screening of Combinatorial Peptide Libraries by Spatially Ordered Beads Immobilized on Conventional Glass Slides
- Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracyScreening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.…
Autor*innen: | Timm Schwaar, Maike Lettow, Dario Remmler, H. G. Börner, Michael G. WellerORCiD |
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Dokumenttyp: | Zeitschriftenartikel |
Veröffentlichungsform: | Verlagsliteratur |
Sprache: | Englisch |
Titel des übergeordneten Werkes (Englisch): | High-Throughput |
Jahr der Erstveröffentlichung: | 2019 |
Organisationseinheit der BAM: | 1 Analytische Chemie; Referenzmaterialien |
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik | |
Veröffentlichende Institution: | Bundesanstalt für Materialforschung und -prüfung (BAM) |
Verlag: | MDPI |
Verlagsort: | Basel |
Jahrgang/Band: | 8 |
Ausgabe/Heft: | 2 |
Erste Seite: | 1 |
Letzte Seite: | 15 |
DDC-Klassifikation: | Naturwissenschaften und Mathematik / Chemie / Analytische Chemie |
Freie Schlagwörter: | Array; Binder; Biochip; HTS; Lab-on-a-Chip; Ladder sequencing; MALDI; Mass spectrometry; Peptide library; Pharmaceutical; Screening; Target |
Themenfelder/Aktivitätsfelder der BAM: | Chemie und Prozesstechnik |
Chemie und Prozesstechnik / Chemische Charakterisierung und Spurenanalytik | |
DOI: | 10.3390/ht8020011 |
URN: | urn:nbn:de:kobv:b43-478973 |
URL: | https://www.mdpi.com/2571-5135/8/2/11 |
Verfügbarkeit des Dokuments: | Datei für die Öffentlichkeit verfügbar ("Open Access") |
Lizenz (Deutsch): | Creative Commons - CC BY - Namensnennung 4.0 International |
Datum der Freischaltung: | 08.05.2019 |
Referierte Publikation: | Ja |
Datum der Eintragung als referierte Publikation: | 13.05.2019 |
Schriftenreihen ohne Nummerierung: | Wissenschaftliche Artikel der BAM |