Enzymes, Immobilized; Kinetics; Ion Mobility Spectrometry/methods; Mass Spectrometry/methods; Enzymes, Immobilized/chemistry; Enzymes, Immobilized/metabolism; Enzymatic reaction; Flow chemistry; Forward designs; In-vitro; Ion Mobility; Ion mobility-mass spectrometry; Online monitoring; Reaction network; Systematic exploration; Ion Mobility Spectrometry; Mass Spectrometry; Catalysis; Chemistry (all); Biochemistry
Abstract :
[en] The forward design of in vitro enzymatic reaction networks (ERNs) requires a detailed analysis of network kinetics and potentially hidden interactions between the substrates and enzymes. Although flow chemistry allows for a systematic exploration of how the networks adapt to continuously changing conditions, the analysis of the reaction products is often a bottleneck. Here, we report on the interface between a continuous stirred-tank reactor, in which an immobilized enzymatic network made of 12 enzymes is compartmentalized, and an ion mobility-mass spectrometer. Feeding uniformly 13C-labeled inputs to the enzymatic network generates all isotopically labeled reaction intermediates and products, which are individually detected by ion mobility-mass spectrometry (IMS-MS) based on their mass-to-charge ratios and inverse ion mobilities. The metabolic flux can be continuously and quantitatively monitored by diluting the ERN output with nonlabeled standards of known concentrations. The real-time quantitative data obtained by IMS-MS are then harnessed to train a model of network kinetics, which proves sufficiently predictive to control the ERN output after a single optimally designed experiment. The high resolution of the time-course data provided by this approach is an important stepping stone to design and control sizable and intricate ERNs.
Disciplines :
Chemistry
Author, co-author :
DUEZ, Quentin ✱; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
van de Wiel, Jeroen ✱; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
van Sluijs, Bob; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
Ghosh, Souvik ; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
Baltussen, Mathieu G. ; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
Derks, Max T. G. M.; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
Roithová, Jana ; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
Huck, Wilhelm T. S. ; Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen 6525 AJ, The Netherlands
✱ These authors have contributed equally to this work.
Language :
English
Title :
Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility-Mass Spectrometry.
European Research Council Nederlandse Organisatie voor Wetenschappelijk Onderzoek
Funding text :
This project is funded by the European Research Council (ERC) under the European Union\u2019s Horizon 2020 research and innovation programme (ERC Adv. grant Life-Inspired, grant agreement no. 833466 and ERC PoC grant OptiPlex, grant agreement no. 101069237) and by the Dutch Research Council (NWO\u2500OCENW.KLEIN.348).
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