High‐Throughput Label‐Free Enzymatic Assays Using Desorption Electrospray‐Ionization Mass Spectrometry

High‐throughput (HT) enzymatic assays, which typically rely on labeled compounds and plate readers, are important for drug discovery. Mass spectrometry (MS) provides an alternative method of performing HT label‐free assays. Here we demonstrate the use of a HT platform based on desorption electrospra...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2020-11, Vol.59 (46), p.20459-20464
Main Authors: Morato, Nicolás M., Holden, Dylan T., Cooks, R. Graham
Format: Article
Language:English
Subjects:
Acetylcholinesterase
analytical methods
Assaying
Bioassays
Biological Assay
Calibration
Desorption
Drug discovery
Drug Discovery - methods
Electrospraying
enzymes
Enzymes - metabolism
Enzymology
high-throughput screening
High-Throughput Screening Assays - methods
Ionization
Ions
label-free
Mass spectrometry
Mass spectroscopy
Scientific imaging
Spectrometry, Mass, Electrospray Ionization - methods
Spectroscopy
Substrate Specificity
Substrates
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Summary:High‐throughput (HT) enzymatic assays, which typically rely on labeled compounds and plate readers, are important for drug discovery. Mass spectrometry (MS) provides an alternative method of performing HT label‐free assays. Here we demonstrate the use of a HT platform based on desorption electrospray ionization (DESI) MS for the label‐free study of enzymatic reactions directly from the bioassay matrix with an effective analysis time of 0.3 s per sample. This system allows for thorough analysis of the enzymatic process through monitoring of its substrate and product after an external calibration. We show the platform capabilities by an in‐depth study of the acetylcholinesterase assay, including kinetic parameter determination, rapid inhibitor screening, and further characterization of positive hits (that is, IC50 and Ki), as well as inhibition–reactivation assays. We anticipate that the expansion of this platform has high potential impact in label‐free enzymology as well as in drug discovery. High‐throughput label‐free enzymatic assays are carried out using an automated platform based on desorption electrospray ionization mass spectrometry. Direct analysis from complex bioassay matrices using nanogram amounts of material in less than 1 s per sample is achieved. Kinetic determinations, inhibitor screening and characterization, as well as inhibition–reactivation studies are all demonstrated for the acetylcholinesterase system.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202009598