scholarly journals High-Throughput Peptide Derivatization toward Supramolecular Diversification in Microtiter Plates

ACS Nano ◽  
2021 ◽  
Author(s):  
Yiyang Lin ◽  
Matthew Penna ◽  
Christopher D. Spicer ◽  
Stuart G. Higgins ◽  
Amy Gelmi ◽  
...  
Keyword(s):  
High Throughput ◽  
Microtiter Plates ◽  
Peptide Derivatization

High-Throughput System for Screening of Cephalosporin C High-Yield Strain by 48-Deep-Well Microtiter Plates

2013 ◽  
Vol 169 (5) ◽  
pp. 1683-1695 ◽  
Author(s):  
Jun Tan ◽  
Ju Chu ◽  
Yuyou Hao ◽  
Yuanxin Guo ◽  
Yingping Zhuang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Yield ◽  
Cephalosporin C ◽  
Yield Strain ◽  
Deep Well ◽  
Microtiter Plates

scholarly journals A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anna Gärtner ◽  
Anna Joëlle Ruff ◽  
Ulrich Schwaneberg
Keyword(s):  
Capillary Electrophoresis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Product Formation ◽  
Resonance Spectroscopy ◽  
P450 Bm3 ◽  
Microtiter Plates ◽  
Main Challenge ◽  
Total Turnover ◽  
Screening Systems

Abstract The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.

A high-throughput system for two-hybrid screening based on growth curve analysis in microtiter plates

2003 ◽  
Vol 316 (2) ◽  
pp. 171-174 ◽  
Author(s):  
Claudia Diaz-Camino ◽  
Eddy P. Risseeuw ◽  
Enwu Liu ◽  
William L. Crosby
Keyword(s):  
High Throughput ◽  
Growth Curve ◽  
Curve Analysis ◽  
Growth Curve Analysis ◽  
Microtiter Plates ◽  
Two Hybrid ◽  
Hybrid Screening

scholarly journals Fluorescence Polarization Assays for High Throughput Screening of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (3) ◽  
pp. 159-167 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Rank Order ◽  
Drug Binding ◽  
G Protein Coupled Receptors ◽  
Microtiter Plates ◽  
High Throughput Drug Screening ◽  
G Protein Coupled ◽  
Speed Of Analysis

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to ['251]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 tiM. These attributes suggest that accurate assessment of drug binding can be obtained.

Development of a high-throughput assay for measuring lipase activity using natural triacylglycerols coated on microtiter plates

The Analyst ◽  
2013 ◽  
Vol 138 (18) ◽  
pp. 5230 ◽  
Author(s):  
Carole Serveau-Avesque ◽  
Robert Verger ◽  
Jorge A. Rodriguez ◽  
Abdelkarim Abousalham
Keyword(s):  
High Throughput ◽  
Lipase Activity ◽  
Microtiter Plates ◽  
High Throughput Assay

scholarly journals High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

2015 ◽  
Vol 21 (3) ◽  
pp. 316-322 ◽  
Author(s):  
Mathew Hanson ◽  
Lorne D. Jordan ◽  
Yan Shipelskiy ◽  
Salete M. Newton ◽  
Phillip E. Klebba
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
High Throughput Screening ◽  
Iron Transport ◽  
Iron Acquisition ◽  
Metabolic Inhibitors ◽  
Screening Assay ◽  
Gram Negative ◽  
Microtiter Plates ◽  
High Throughput Screening Assay

The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8–1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria.

scholarly journals Rapid Methods for High-Throughput Detection of Sulfoxides

2009 ◽  
Vol 75 (14) ◽  
pp. 4711-4719 ◽  
Author(s):  
Janna Shainsky ◽  
Netta-Lee Derry ◽  
Yael Leichtmann-Bardoogo ◽  
Thomas K. Wood ◽  
Ayelet Fishman
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Selective Inhibition ◽  
Saturation Mutagenesis ◽  
Screening Methods ◽  
Acid Activation ◽  
Special Equipment ◽  
Whole Cell ◽  
Microtiter Plates

ABSTRACT Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.

Acoustic Ejection/Full-Scan Mass Spectrometry Analysis for High-Throughput Compound Quality Control

2020 ◽  
pp. 247263032096762
Author(s):  
Jun Zhang ◽  
Yong Zhang ◽  
Chang Liu ◽  
Tom Covey ◽  
Julia Nielsen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Data Processing ◽  
High Throughput ◽  
Data Extraction ◽  
Mass Spectrometry Analysis ◽  
High Throughput Analysis ◽  
Spectrometry Analysis ◽  
Microtiter Plates ◽  
Full Scan

High-throughput analysis of compound dissolved in DMSO and arrayed in multiwell plates for quality control (QC) purposes has widespread utility in drug discovery, ranging from the QC of assay-ready plates dispatched by compound management, to compound integrity check in the screening collection, to reaction monitoring of chemical syntheses in microtiter plates. Due to the large number of samples (thousands per batch) involved, these workflows can put a significant burden on the liquid chromatography–mass spectrometry (LC-MS) platform typically used. To achieve the required speed of seconds per sample, several chromatography-free MS approaches have previously been used with mixed results. In this study, we demonstrated the feasibility of acoustic ejection–mass spectrometry (AE-MS) in full-scan mode for high-throughput compound QC in miniaturized formats, featuring direct, contactless liquid sampling, minimal sample consumption, and ultrafast analytical speed. The sample consumption and analysis time by AE-MS represent, respectively, a 1000-fold and 30-fold reduction compared with LC-MS. In qualitative QC, AE-MS generated comparable results to conventional LC-MS in identifying the presence and absence of expected compounds. AE-MS also demonstrated its utility in relative quantifications of the same compound in serial dilution plates, or substrate in chemical synthesis. To facilitate the processing of a large amount of data generated by AE-MS, we have developed a data processing platform using commercially available tools. The platform demonstrated fast and straightforward data extraction, reviewing, and reporting, thus eliminating the need for the development of custom data processing tools. The overall AE-MS workflow has effectively eliminated the analytical bottleneck in the high-throughput compound QC work stream.

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