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Development of an HPLC-HRMS method for quality control of peptide pools

  • Synthetic peptide pools are used in antigen-specific T-cell assays, which are an important part in vaccine and immunotherapeutic clinical trials. As the analytical characterization is challenging due to the similarity of the single peptides or is expensive due to isotope labeled standards, usually only a pre-characterization of the single peptides is performed. However, a regular quality control of the peptide mix would be highly desirable. Therefore, a cost-efficient high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for quality control of a model peptide pool is developed. Peptides were synthesized using peptides&elephants proprietary libraries of individual peptides (LIPS) technology and purified by reversed-phase chromatography to > 90% each. The lyophilized single peptides were combined to a model peptide pool and analyzed by reversed-phase high-performance capillary liquid chromatography coupled to an orbitrap mass spectrometer.Synthetic peptide pools are used in antigen-specific T-cell assays, which are an important part in vaccine and immunotherapeutic clinical trials. As the analytical characterization is challenging due to the similarity of the single peptides or is expensive due to isotope labeled standards, usually only a pre-characterization of the single peptides is performed. However, a regular quality control of the peptide mix would be highly desirable. Therefore, a cost-efficient high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for quality control of a model peptide pool is developed. Peptides were synthesized using peptides&elephants proprietary libraries of individual peptides (LIPS) technology and purified by reversed-phase chromatography to > 90% each. The lyophilized single peptides were combined to a model peptide pool and analyzed by reversed-phase high-performance capillary liquid chromatography coupled to an orbitrap mass spectrometer. Separation was performed on a capillary reversed phase column (2 μm, ID x L 300 μm x 150 mm) with a linear gradient of acetonitrile + 0,05% trifluoroacetic acid. For the separation of a model peptide pool the additive, additive concentration, the gradient elution and the temperature were optimized. Different quantification approaches were tested. Identification was performed by high resolution mass spectrometry in which extracted ion chromatograms (XIC) were used to confirm exact masses. In line with the development of a new cost-efficient quality control method for the separation and identification of complex synthetic peptide pools, varied HPLC parameters highlighted their influence on chromatographic resolution and peptides were identified with high mass accuracy.zeige mehrzeige weniger

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Metadaten
Autor*innen:Gaby Bosc-Bierne
Dokumenttyp:Vortrag
Veröffentlichungsform:Präsentation
Sprache:Englisch
Jahr der Erstveröffentlichung:2018
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:LC-MS; Peptide pools; Quality control
Themenfelder/Aktivitätsfelder der BAM:Chemie und Prozesstechnik
Veranstaltung:8th Austrian Peptide Symposium
Veranstaltungsort:Salzburg, Austria
Beginndatum der Veranstaltung:13.12.2018
Enddatum der Veranstaltung:13.12.2018
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:18.12.2018
Referierte Publikation:Nein
Eingeladener Vortrag:Nein
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