High-throughput mapping of CoA metabolites by SAMDI-MS to optimize the cell-free biosynthesis of HMG-CoA

O’Kane, Patrick T.; Dudley, Quentin M.; McMillan, Aislinn K.; Jewett, Michael C.; Mrksich, Milan

doi:10.1126/sciadv.aaw9180

Cited by 43 publications

(38 citation statements)

References 49 publications

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“…This study reports the first label-free and high-throughput assay to measure coronavirus 3CLpro activity and opens avenues for improved drug discovery efforts to find small molecule inhibitors to treat COVID-19. The SAMDI-MS technology has been reported to characterize dozens of biochemical activities including post-translational modifying enzymes ( Gurard-Levin et al, 2011 ; Swalm et al, 2014 ; Patel et al, 2015 ; Wigle et al, 2015 ; Szymczak et al, 2018 ; O'Kane et al, 2019 ), RNA-modifying proteins ( Buker et al, 2020 ), and proteases ( Min et al, 2004 ) and can be applied to virtually any enzymatic activity. While the FRET-based assay format is convenient and can easily be adopted by standard biochemistry labs, this study demonstrates that SAMDI-MS offers distinct advantages over label-dependent FRET that were critical in developing the SARS-CoV-2 3CLpro assay ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry

Gurard-Levin¹,

Liu²,

Jekle³

et al. 2020

Antiviral Research

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC 50 = 0.060 μM), calpain inhibitors II and XII (IC 50 ~20–25 μM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human β-coronavirus OC-43 or SARS-CoV-2 in vitro , but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2.

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Section: Discussionmentioning

confidence: 99%

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry

Gurard-Levin¹,

Liu²,

Jekle³

et al. 2020

Antiviral Research

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“…Recently, cell‐free systems have emerged as an attractive and powerful platform for biomanufacturing (Bundy et al, 2018; Carlson, Gan, Hodgman, & Jewett, 2012; Dudley, Karim, & Jewett, 2015; Li, Zhang, & Liu, 2018; Liu, Zhang, Chen, & Li, 2019; Silverman, Karim, & Jewett, 2020; Swartz, 2018). In this context, cell‐free protein synthesis (CFPS) plays a core role in the production of various proteins in vitro (Jaroentomeechai et al, 2018; Kwon et al, 2013; Li et al, 2016; Lu, Welsh, & Swartz, 2014; Martin et al, 2018; McNerney et al, 2019; O'Kane, Dudley, McMillan, Jewett, & Mrksich, 2019; Wilding et al, 2019). Because of the absence of cell walls, cell‐free systems bypass mass transfer limitations and are more tolerant of toxic substrates, intermediates, and products than living microbial cells.…”

Section: Figurementioning

confidence: 99%

Cell‐free protein synthesis enables one‐pot cascade biotransformation in an aqueous‐organic biphasic system

Liu

Jewett

et al. 2020

Biotech & Bioengineering

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Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueousorganic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our cell-free approach will make a possible cost-effective, high-yielding synthesis of valuable chemicals.

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“…Currently, existing TOF mass spectrometers experimentally achieve a mass resolution of up to 300,000 in the full mass range in biochemical applications, or up to 600,000 in a limited mass range in nuclear physics research . Compared with this, the aberration limit achieved in the proposed analyzer (excluding the turnaround time) with very low ion emission (1% energy dispersion) is estimated by us as 30,000.…”

Section: Results and Discussion – Resolution Estimations Of Multi‐casmentioning

confidence: 98%

Time‐of‐flight mass spectrometers based on a wedge‐shaped electrostatic mirror with a two‐dimensional field

Spivak-Lavrov

Baisanov²,

Yakushev³

et al. 2020

Rapid Comm Mass Spectrometry

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Rationale: For most of the last two decades, a considerable effort has been made towards improving time-of-flight mass spectrometry (TOF MS), which has become an irreplaceable instrumental platform for the purposes of performing analytical measurements in life sciences, such as molecular biology, proteomics, medicine, etc.This can primarily be attributed to the ability of TOF MS to rapidly detect and identify nearly any targeted chemical trace with both high precision and accuracy.However, multi-span TOF MS experiments are limited due to aberrations arising from multiple reflection; our proposed scheme will minimize these aberrations. Methods:The inhomogeneous accelerating field is generated without using meshes by changing the potentials on the electrodes of the mirror. The ions are extracted from the ion source by short impulse activation of the accelerating electric field.Since the ions are extracted from various points of the source, even ions with identical masses acquire different velocities during acceleration. Results:We have shown that the "rear" ions of the packet catch up with the "front" ions, and packets of ions with identical masses are compressed in the direction of their movement. It is concluded that, by placing the detector in a plane with the greatest compression of ion packets, an enhanced performance of a time-of-flight mass spectrometer is achieved. Conclusions:We have shown that effective spatial-temporal focusing allows a small mass spectrometer to achieve high resolution and sensitivity. We also propose and numerically evaluate a new platform for designing multi-stage and multireflective time-of-flight analyzers with wedge-shaped mirrors. We applied the simulation results to the modernization of old equipment and showed that by simply replacing the electrostatic mirror with an optimized one, a significant increase in the analyzing power can be achieved.

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High-throughput mapping of CoA metabolites by SAMDI-MS to optimize the cell-free biosynthesis of HMG-CoA

Cited by 43 publications

References 49 publications

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry

Cell‐free protein synthesis enables one‐pot cascade biotransformation in an aqueous‐organic biphasic system

Time‐of‐flight mass spectrometers based on a wedge‐shaped electrostatic mirror with a two‐dimensional field

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