spectra of substrates/products at concentrations ranging from
1 to 10 μM. Use of a surfactant can influence the droplet
formation in the acoustic event, which may promote the ionization process but can also cause ion suppression, hence reducing detection.
4. All data have been collected using a Waters Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer fitted with a
universal ion source. One of the features of the Waters mass
spectrometer employed is the ability to enhance sensitivity for a
small mass range, nominally 200–300 Da.
5. For example, MS acquisition from 500 to 700 Da with a scan
time of 100 ms reduces the amount of data collected and
produces enough data points across a well ejection.
6. If salts are required for the enzyme reaction, the MS interference problem can be solved in two ways. The simplest is by
adding a large volume of stop reagent at the end of the assay, as
this dilutes the salts down prior to the AMI-MS read. Alternatively, the salts can be precipitated out of the solution in the
stop step. For example, MgCl 2 can be precipitated by using the
appropriate amount of ammonium phosphate in the assay
buffer, which precipitates the salt on addition of ammonium
hydroxide during addition of the stop reagent.
7. Users should not see any difference between using assay-ready
plates and plates prepared using more conventional methods
where a solution of compound is diluted stepwise prior to
adding to the plate. It is important that the DMSO concentration is 1% (v/v) or lower as DMSO in higher concentrations is
potentially inhibitory.
8. It is important to consider carefully where to place control wells
on the plates. It is quite common to see control wells placed on
the outermost columns, but this is not recommended as these
wells will be affected by any edge effect, which in turn will lead
to incorrect normalization of the data. Control wells are placed
in the middle of the plate to minimize any edge effects. Ideally,
controls could be dispersed across more of the plate, but this is
difficult to achieve with more traditional liquid handling equipment and adds complexity, although these layouts are
employed for some concentration-response screens.
9. A volume of 50 μL in standard 384-well plates is necessary as
this improves firing in the AMI-MS. A 10 μL + 10 μL addition
for the enzyme and substrate is generally necessary to ensure
the two components mix effectively due to the large surface
area of the well base. To reduce reagent costs, these ratios could
be altered. It is possible to use lower volume additions, e.g.,
2 μL of enzyme plus 2 μL of substrate, if a centrifuge step is
added. This allows mixing of the components. Following this, a
228
Helen Plant et al.
1 to 10 μM. Use of a surfactant can influence the droplet
formation in the acoustic event, which may promote the ionization process but can also cause ion suppression, hence reducing detection.
4. All data have been collected using a Waters Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer fitted with a
universal ion source. One of the features of the Waters mass
spectrometer employed is the ability to enhance sensitivity for a
small mass range, nominally 200–300 Da.
5. For example, MS acquisition from 500 to 700 Da with a scan
time of 100 ms reduces the amount of data collected and
produces enough data points across a well ejection.
6. If salts are required for the enzyme reaction, the MS interference problem can be solved in two ways. The simplest is by
adding a large volume of stop reagent at the end of the assay, as
this dilutes the salts down prior to the AMI-MS read. Alternatively, the salts can be precipitated out of the solution in the
stop step. For example, MgCl 2 can be precipitated by using the
appropriate amount of ammonium phosphate in the assay
buffer, which precipitates the salt on addition of ammonium
hydroxide during addition of the stop reagent.
7. Users should not see any difference between using assay-ready
plates and plates prepared using more conventional methods
where a solution of compound is diluted stepwise prior to
adding to the plate. It is important that the DMSO concentration is 1% (v/v) or lower as DMSO in higher concentrations is
potentially inhibitory.
8. It is important to consider carefully where to place control wells
on the plates. It is quite common to see control wells placed on
the outermost columns, but this is not recommended as these
wells will be affected by any edge effect, which in turn will lead
to incorrect normalization of the data. Control wells are placed
in the middle of the plate to minimize any edge effects. Ideally,
controls could be dispersed across more of the plate, but this is
difficult to achieve with more traditional liquid handling equipment and adds complexity, although these layouts are
employed for some concentration-response screens.
9. A volume of 50 μL in standard 384-well plates is necessary as
this improves firing in the AMI-MS. A 10 μL + 10 μL addition
for the enzyme and substrate is generally necessary to ensure
the two components mix effectively due to the large surface
area of the well base. To reduce reagent costs, these ratios could
be altered. It is possible to use lower volume additions, e.g.,
2 μL of enzyme plus 2 μL of substrate, if a centrifuge step is
added. This allows mixing of the components. Following this, a
228
Helen Plant et al.