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 ORCID: https://orcid.org/0000-0002-8035-4719, Somsen, Govert W and Kool, Jeroen (2019) 'Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation.'. Analytical Chemistry, Vol 91, Issue 16, pp. 10458-10466.
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Abstract
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.
Item Type: | Article |
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Subjects: | QU Biochemistry > QU 4 General works QV Pharmacology > Hematologic Agents > QV 190 Drugs affecting blood coagulation QV Pharmacology > Hematologic Agents > QV 195 Hemostatics. Coagulants QV Pharmacology > Drug Standardization. Pharmacognosy. Medicinal Plants > QV 766 Medicinal plants (General) QV Pharmacology > Drug Standardization. Pharmacognosy. Medicinal Plants > QV 771 Standardization and evaluation of drugs |
Faculty: Department: | Biological Sciences > Department of Tropical Disease Biology |
Digital Object Identifer (DOI): | https://doi.org/10.1021/acs.analchem.9b01261 |
Depositing User: | Mary Creegan |
Date Deposited: | 12 Aug 2019 08:37 |
Last Modified: | 06 Sep 2019 14:08 |
URI: | https://archive.lstmed.ac.uk/id/eprint/11389 |
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