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High Throughput Screening with SAMDI Mass Spectrometry for Directed Evolution

Pluchinsky, Adam J. and Wackelin, Daniel J. and Huang, Xiongyi and Arnold, Frances H. and Mrksich, Milan (2020) High Throughput Screening with SAMDI Mass Spectrometry for Directed Evolution. Journal of the American Chemical Society, 142 (47). pp. 19804-19808. ISSN 0002-7863. https://resolver.caltech.edu/CaltechAUTHORS:20201111-125109727

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Abstract

Advances in directed evolution have led to an exploration of new and important chemical transformations; however, many of these efforts still rely on the use of low-throughput chromatography-based screening methods. We present a high-throughput strategy for screening libraries of enzyme variants for improved activity. Unpurified reaction products are immobilized to a self-assembled monolayer and analyzed by mass spectrometry, allowing for direct evaluation of thousands of variants in under an hour. The method was demonstrated with libraries of randomly mutated cytochrome P411 variants to identify improved catalysts for C–H alkylation. The technique may be tailored to evolve enzymatic activity for a variety of transformations where higher throughput is needed.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/jacs.0c07828DOIArticle
ORCID:
AuthorORCID
Huang, Xiongyi0000-0001-7156-8881
Arnold, Frances H.0000-0002-4027-364X
Mrksich, Milan0000-0002-4964-796X
Additional Information:© 2020 American Chemical Society. Received: July 20, 2020; Published: November 11, 2020. We thank Dr. Ruijie Zhang for initial discussions and Dr. Sabine-Brinkmann-Chen for critical review of the manuscript. A.J.P. and D.J.W. are supported by the National Science Foundation Graduate Research Fellowship under Grant Nos. DGE-1842165 and DGE-174530. Research reported in this publication was supported by the Department of Defense, Defense Threat Reduction Agency, under award HDTRA1-15-1-0052 (to M.M.) and by the US Army Research Office Institute for Collaborative Biotechnologies cooperative agreement W911NF-19-2-0026 (to F.H.A. and X.H.). Author Contributions: A.J.P. and D.J.W. contributed equally to this work. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
NSF Graduate Research FellowshipDGE-1842165
NSF Graduate Research FellowshipDGE-174530
Defense Threat Reduction Agency (DTRA)HDTRA1-15-1-0052
Army Research Office (ARO)W911NF-19-2-0026
Issue or Number:47
Record Number:CaltechAUTHORS:20201111-125109727
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201111-125109727
Official Citation:High Throughput Screening with SAMDI Mass Spectrometry for Directed Evolution. Adam J. Pluchinsky, Daniel J. Wackelin, Xiongyi Huang, Frances H. Arnold, and Milan Mrksich. Journal of the American Chemical Society 2020 142 (47), 19804-19808; DOI: 10.1021/jacs.0c07828
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106617
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:11 Nov 2020 22:19
Last Modified:25 Nov 2020 17:42

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