| Title: | The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases |
| Author(s): | Di Girolamo A; Pedrotti M; Koot A; Verstappen F; van Houwelingen A; Vossen C; Bouwmeester H; de Ridder D; Beekwilder J; |
| Address: | "Laboratory of Plant Physiology, Department of Plant Sciences, Wageningen University, Wageningen, The Netherlands. Post-Harvest Technology Group, Wageningen Food & Biobased Research, Wageningen Research, Wageningen, The Netherlands. Authenticity & Nutrients Group, Wageningen Food Safety Research, Wageningen Research, Wageningen, The Netherlands. Bioscience, Wageningen Plant Research, Wageningen University, Wageningen, The Netherlands. Isobionics BV, Geleen, The Netherlands. Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. Bioinformatics Group, Department of Plant Sciences, Wageningen University, Wageningen, The Netherlands" |
| ISSN/ISBN: | 1096-9888 (Electronic) 1076-5174 (Linking) |
| Abstract: | "In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR-MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC-MS)-based methods, PTR-MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR-MS-based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR-Qi-ToF-MS were compared with GC-MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR-Qi-ToF-MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC-MS analysis. This study provides a first basis for the application of rapid PTR-Qi-ToF-MS detection, in combination with multi-well formats, in HTS screening methods for the selection of highly productive terpene synthases" |
| Keywords: | *Protons High-Throughput Screening Assays Mass Spectrometry/methods Terpenes *Volatile Organic Compounds/analysis Ptr-ms VOCs high throughput screening sesquiterpene valencene; |
| Notes: | "MedlineDi Girolamo, Alice Pedrotti, Michele Koot, Alex Verstappen, Francel van Houwelingen, Adele Vossen, Celina Bouwmeester, Harro de Ridder, Dick Beekwilder, Jules eng TTW 15043/Netherlands Organisation for Scientific Research (NWO)/ England 2023/06/01 J Mass Spectrom. 2023 Jun; 58(6):e4951. doi: 10.1002/jms.4951" |
