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Versatile synthesis of probes for high-throughput enzyme activity screening |
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| Authors: | Tristan de Rond Pamela Peralta-Yahya Xiaoliang Cheng Trent R Northen Jay D Keasling |
| Institution: | 1. Department of Chemistry, University of California, Berkeley, CA, 94720, USA 2. California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA 3. Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA, 94608, USA 4. Department of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, GA, 30332, USA 5. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA 6. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA 7. Chemical and Biomolecular Engineering and Bioengineering Departments, University of California, Berkeley, CA, 94720, USA |
| Abstract: | Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination. The Nimzyme high-throughput enzyme activity assay allows for the detection of enzyme activity in cell lysate. Fluorous interactions between a specialized substrate probe and a nanostructure-initiator mass spectrometry surface allow for in situ cleanup and the subsequent collection of unambiguous mass spectra. One of the main hurdles that prevents the widespread adoption of this technology is the need to chemically synthesize the required probes. Here, we present a simplified route to derive Nimzyme probes from a wide variety of biologically interesting substrates. |
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