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Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation
[Image: see text] High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and s...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2019
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706796/ https://www.ncbi.nlm.nih.gov/pubmed/31373797 http://dx.doi.org/10.1021/acs.analchem.9b01261 |
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author | Zietek, Barbara M. Still, Kristina B. M. Jaschusch, Kevin Bruyneel, Ben Ariese, Freek Brouwer, Tinco J. F. Luger, Matthijs Limburg, Rob J. Rosier, Joost C. v. Iperen, Dick J. Casewell, Nicholas R. Somsen, Govert W. Kool, Jeroen |
author_facet | Zietek, Barbara M. Still, Kristina B. M. Jaschusch, Kevin Bruyneel, Ben Ariese, Freek Brouwer, Tinco J. F. Luger, Matthijs Limburg, Rob J. Rosier, Joost C. v. Iperen, Dick J. Casewell, Nicholas R. Somsen, Govert W. Kool, Jeroen |
author_sort | Zietek, Barbara M. |
collection | PubMed |
description | [Image: see text] High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases. |
format | Online Article Text |
id | pubmed-6706796 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-67067962019-08-26 Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation Zietek, Barbara M. Still, Kristina B. M. Jaschusch, Kevin Bruyneel, Ben Ariese, Freek Brouwer, Tinco J. F. Luger, Matthijs Limburg, Rob J. Rosier, Joost C. v. Iperen, Dick J. Casewell, Nicholas R. Somsen, Govert W. Kool, Jeroen Anal Chem [Image: see text] High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases. American Chemical Society 2019-08-02 2019-08-20 /pmc/articles/PMC6706796/ /pubmed/31373797 http://dx.doi.org/10.1021/acs.analchem.9b01261 Text en Copyright © 2019 American Chemical Society This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License (http://pubs.acs.org/page/policy/authorchoice_ccbyncnd_termsofuse.html) , which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. |
spellingShingle | Zietek, Barbara M. Still, Kristina B. M. Jaschusch, Kevin Bruyneel, Ben Ariese, Freek Brouwer, Tinco J. F. Luger, Matthijs Limburg, Rob J. Rosier, Joost C. v. Iperen, Dick J. Casewell, Nicholas R. Somsen, Govert W. Kool, Jeroen Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation |
title | Bioactivity Profiling of Small-Volume Samples by Nano
Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution
Fractionation |
title_full | Bioactivity Profiling of Small-Volume Samples by Nano
Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution
Fractionation |
title_fullStr | Bioactivity Profiling of Small-Volume Samples by Nano
Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution
Fractionation |
title_full_unstemmed | Bioactivity Profiling of Small-Volume Samples by Nano
Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution
Fractionation |
title_short | Bioactivity Profiling of Small-Volume Samples by Nano
Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution
Fractionation |
title_sort | bioactivity profiling of small-volume samples by nano
liquid chromatography coupled to microarray bioassaying using high-resolution
fractionation |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706796/ https://www.ncbi.nlm.nih.gov/pubmed/31373797 http://dx.doi.org/10.1021/acs.analchem.9b01261 |
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