Chapter 9
The Use of Acoustic Mist Ionization Mass Spectrometry
for High-Throughput Screening
Helen Plant, David Murray, Hannah Semple, Gareth Davies, Ian Sinclair,
and Geoffrey A. Holdgate
Abstract
It is clear from the analysis of the distribution of approved drug targets that enzymes continue to be a major
target class for the pharmaceutical industry. The application of high-throughput screens designed to
monitor the activity of these enzyme targets, and the ability of test compounds to modulate this activity,
is still the predominant hit finding approach in the industry. The widespread use of enzyme activity-based
screens has led to the development of several useful guidelines for the development and validation of robust
and reliable assays. Key learnings for the development, validation, and implementation of acoustic mist
ionization mass spectrometry for high-throughput enzyme assays are described.
Key words Mass spectrometry, Acoustic mist ionization, High-throughput screening, 384-well,
Enzyme, Substrate, Label free, Plate reader
1 Introduction
The purpose of high-throughput enzyme activity screening is to
allow monitoring of any particular transformation of substrate to
product and allow detection of molecules that modulate the target
enzyme activity. In general, high-throughput activity assays for
enzymes have been developed using two main approaches. These
are the detection of substrate depletion and the detection of product formation. The development of sensitive enzyme assays suitable
for high-throughput screening (HTS) requires consideration of
several factors: the biochemical reagents required, choice of technology, and the desired mode(s) of action to be identified. Reagent
consideration encompasses the identification of relevant enzyme
and substrate forms, cofactors, buffers, and additives essential to
build a robust biological system. The choice of detection technology will involve aligning the biological system with the appropriate
assay methodology to provide acceptable sensitivity, throughput,
and cost. The desired modes of action that the assay should identify
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
217
The Use of Acoustic Mist Ionization Mass Spectrometry
for High-Throughput Screening
Helen Plant, David Murray, Hannah Semple, Gareth Davies, Ian Sinclair,
and Geoffrey A. Holdgate
Abstract
It is clear from the analysis of the distribution of approved drug targets that enzymes continue to be a major
target class for the pharmaceutical industry. The application of high-throughput screens designed to
monitor the activity of these enzyme targets, and the ability of test compounds to modulate this activity,
is still the predominant hit finding approach in the industry. The widespread use of enzyme activity-based
screens has led to the development of several useful guidelines for the development and validation of robust
and reliable assays. Key learnings for the development, validation, and implementation of acoustic mist
ionization mass spectrometry for high-throughput enzyme assays are described.
Key words Mass spectrometry, Acoustic mist ionization, High-throughput screening, 384-well,
Enzyme, Substrate, Label free, Plate reader
1 Introduction
The purpose of high-throughput enzyme activity screening is to
allow monitoring of any particular transformation of substrate to
product and allow detection of molecules that modulate the target
enzyme activity. In general, high-throughput activity assays for
enzymes have been developed using two main approaches. These
are the detection of substrate depletion and the detection of product formation. The development of sensitive enzyme assays suitable
for high-throughput screening (HTS) requires consideration of
several factors: the biochemical reagents required, choice of technology, and the desired mode(s) of action to be identified. Reagent
consideration encompasses the identification of relevant enzyme
and substrate forms, cofactors, buffers, and additives essential to
build a robust biological system. The choice of detection technology will involve aligning the biological system with the appropriate
assay methodology to provide acceptable sensitivity, throughput,
and cost. The desired modes of action that the assay should identify
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
217