also must be considered during the assay development phase. Typically, this requires designing biologically relevant assays, where
possible utilizing full-length enzyme and physiological substrates,
in which the concentration of the different enzyme forms present in
the assay is approximately balanced [1, 2].
Historically, many high-throughput enzyme assays have been
based on the use of spectrophotometric measurements, including
absorbance [3], fluorescence [4, 5], or luminescence technologies
[6]. Recently mass spectrometry (MS) [7, 8] has emerged as an
increasingly applied technology for monitoring enzyme reaction
progression. Direct measurement of substrate depletion and product formation using label-free detection, combined with a low
artifact hit rate compared to traditional assays, makes this an attractive screening technology. Simple biochemical assay builds of
enzyme plus substrate also makes MS amenable to automated
assay plate generation. Recent breakthroughs in technology development [9] allowing lower sample volumes combined with higher
processing speeds now make acoustic mist ionization mass spectrometry (AMI-MS) suitable for high-throughput screening of
hundreds of thousands of compounds [10]. This chapter describes
key information and considerations for developing and screening
robust AMI-MS assays at scale.
2 Materials
Acoustic mist ionization mass spectrometry (AMI-MS) is a novel
technology for directly introducing the contents of an assay well
into a mass spectrometer at high-throughput. AMI-MS is a labelfree method preferred for directly measuring the substrate to product conversion achieved by enzyme assays, due to the advantages
afforded in the fidelity of the readout and the greatly reduced
susceptibility to assay technology artifacts. The AMI-MS platform
currently exists only as a prototype instrument, which we have been
developing and using within Hit Discovery department at AstraZeneca and is now used in business-as-usual screening activities.
2.1 Instrumentation
AMI-MS requires that samples are introduced into the mass spectrometer via acoustic dispensing. The current prototype combines a
Waters Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer fitted with a universal ion source coupled to a transducer
from Echo
® 555 integrated with a motorized XY stage (Fig. 1).
2.2 Plates
It is essential to use 384-well polypropylene source microplates that
are Echo
® -qualified, such as 384 PP plates catalog number
P-05525 from Labcyte (see Note 1).
218
Helen Plant et al.
possible utilizing full-length enzyme and physiological substrates,
in which the concentration of the different enzyme forms present in
the assay is approximately balanced [1, 2].
Historically, many high-throughput enzyme assays have been
based on the use of spectrophotometric measurements, including
absorbance [3], fluorescence [4, 5], or luminescence technologies
[6]. Recently mass spectrometry (MS) [7, 8] has emerged as an
increasingly applied technology for monitoring enzyme reaction
progression. Direct measurement of substrate depletion and product formation using label-free detection, combined with a low
artifact hit rate compared to traditional assays, makes this an attractive screening technology. Simple biochemical assay builds of
enzyme plus substrate also makes MS amenable to automated
assay plate generation. Recent breakthroughs in technology development [9] allowing lower sample volumes combined with higher
processing speeds now make acoustic mist ionization mass spectrometry (AMI-MS) suitable for high-throughput screening of
hundreds of thousands of compounds [10]. This chapter describes
key information and considerations for developing and screening
robust AMI-MS assays at scale.
2 Materials
Acoustic mist ionization mass spectrometry (AMI-MS) is a novel
technology for directly introducing the contents of an assay well
into a mass spectrometer at high-throughput. AMI-MS is a labelfree method preferred for directly measuring the substrate to product conversion achieved by enzyme assays, due to the advantages
afforded in the fidelity of the readout and the greatly reduced
susceptibility to assay technology artifacts. The AMI-MS platform
currently exists only as a prototype instrument, which we have been
developing and using within Hit Discovery department at AstraZeneca and is now used in business-as-usual screening activities.
2.1 Instrumentation
AMI-MS requires that samples are introduced into the mass spectrometer via acoustic dispensing. The current prototype combines a
Waters Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer fitted with a universal ion source coupled to a transducer
from Echo
® 555 integrated with a motorized XY stage (Fig. 1).
2.2 Plates
It is essential to use 384-well polypropylene source microplates that
are Echo
® -qualified, such as 384 PP plates catalog number
P-05525 from Labcyte (see Note 1).
218
Helen Plant et al.