the output of a screen. In this way, the setup of the assay should be
well considered and validated to ensure reliability throughout a
screen and produce the desired output. This method will describe
the considerations necessary for setting up an effective AMI-MS
assay for use in HTS.
AMI-MS assays require use of a simple biochemical assay buffer
such as Tris or tricine, as many reagents interfere with signal detection. For example, salts interfere with signal detection as they can
cause ion suppression or reduce the sensitivity of the detection.
Even the acid or base used to set the pH of such buffers can be
important. Inorganic acids or bases such as sodium hydroxide can
have detrimental effects on acoustic firing; hence, the use of ammonium hydroxide as a base and acetic or formic acid is recommended
for setting buffer pH (see Note 6).
Assay conditions should be optimized to ensure that the signal
measured is directly proportional to the rate of reaction. Ensure
that enzyme progress curves and the initial rates generated from
them are measured in the linear phase with respect to time and
enzyme concentration, respectively, and that a proportional
decrease in signal is reflective of inhibition and in no way compromises the sensitivity of the assay. Where possible, the assay should
be run using a substrate concentration(s) around K m , to allow a
balanced probability of detecting all modes of inhibition [11]. Measuring assay parameters such as K m can be technically difficult in
AMI-MS if an internal standard is not available, due to the variability of the data produced (Subheading 3.6). This variability is due
to differences in firing events from well to well; hence, in the
absence of an internal standard, normalization cannot be performed. In this case, K m should be measured under the same
assay conditions but using an alternative detection system, e.g.,
LC-MS, and this value should be used to establish the concentration of the substrate to use in the assay.
Another variable that should be optimized prior to an AMI-MS
HTS is the volume of ejection into the spectrometer. This can affect
the quality of the output data, especially the variability (Table 1). A
larger ejection volume increases the read time and so the final
volume selected will be a compromise between data quality and
throughput.
One key advantage of AMI-MS over other screening technologies is the ability to measure the conversion of multiple substrates
to products in one assay. The advantage of measuring multiple
endpoints is that a choice of substrate to product conversion may
allow multiplexing of the measurement of initial rate for single-step
multisubstrate reactions. It also provides a route for rate measurements for the two half-reactions carried out by enzymes catalyzing
sequential reactions using independent active sites. This dual endpoint assay can be set up by running the assay and then reading the
plates twice in AMI-MS, enhancing for the different masses for the
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Helen Plant et al.
well considered and validated to ensure reliability throughout a
screen and produce the desired output. This method will describe
the considerations necessary for setting up an effective AMI-MS
assay for use in HTS.
AMI-MS assays require use of a simple biochemical assay buffer
such as Tris or tricine, as many reagents interfere with signal detection. For example, salts interfere with signal detection as they can
cause ion suppression or reduce the sensitivity of the detection.
Even the acid or base used to set the pH of such buffers can be
important. Inorganic acids or bases such as sodium hydroxide can
have detrimental effects on acoustic firing; hence, the use of ammonium hydroxide as a base and acetic or formic acid is recommended
for setting buffer pH (see Note 6).
Assay conditions should be optimized to ensure that the signal
measured is directly proportional to the rate of reaction. Ensure
that enzyme progress curves and the initial rates generated from
them are measured in the linear phase with respect to time and
enzyme concentration, respectively, and that a proportional
decrease in signal is reflective of inhibition and in no way compromises the sensitivity of the assay. Where possible, the assay should
be run using a substrate concentration(s) around K m , to allow a
balanced probability of detecting all modes of inhibition [11]. Measuring assay parameters such as K m can be technically difficult in
AMI-MS if an internal standard is not available, due to the variability of the data produced (Subheading 3.6). This variability is due
to differences in firing events from well to well; hence, in the
absence of an internal standard, normalization cannot be performed. In this case, K m should be measured under the same
assay conditions but using an alternative detection system, e.g.,
LC-MS, and this value should be used to establish the concentration of the substrate to use in the assay.
Another variable that should be optimized prior to an AMI-MS
HTS is the volume of ejection into the spectrometer. This can affect
the quality of the output data, especially the variability (Table 1). A
larger ejection volume increases the read time and so the final
volume selected will be a compromise between data quality and
throughput.
One key advantage of AMI-MS over other screening technologies is the ability to measure the conversion of multiple substrates
to products in one assay. The advantage of measuring multiple
endpoints is that a choice of substrate to product conversion may
allow multiplexing of the measurement of initial rate for single-step
multisubstrate reactions. It also provides a route for rate measurements for the two half-reactions carried out by enzymes catalyzing
sequential reactions using independent active sites. This dual endpoint assay can be set up by running the assay and then reading the
plates twice in AMI-MS, enhancing for the different masses for the
222
Helen Plant et al.