stop mixture should be added to give a final volume of 50 μL
for the read. Using a low-volume reaction mixture and having a
large dilution to 50 μL final volume is advantageous when
using salts, as discussed in Note 6. Low-volume 384-well
polypropylene plates are a viable alternative, which would
allow lower volumes of reagents to be used. Currently, these
plates are expensive, so they may only be beneficial if the cost of
reagents outweighs the extra costs of the plates.
10. When storing assay plates to be read or re-read after more than
a day, they should be stored at ambient temperature rather than
cold. Storing assay plates at 4
C appears to reduce the ability to
fire on the MS. If plates are stored cold, they must be allowed
to fully equilibrate to ambient temperature prior to reading.
Plates should be stored in a stack or with a lid on to reduce
evaporation. Water may be added to the plates if the volume
has been reduced by evaporation when in storage.
11. It was decided to mask any well where the measured substrate
value is 0, but other low reading wells are taken through to
confirmation or IC/EC 50 screening to ensure that potential
hits are not missed.
12. Use of an internal standard in any AMI-MS assay greatly
improves ease of analysis as data can be referenced to and
normalized against this standard. Anything that does not interfere with the assay, such as a modified substrate or product or
any derivative of these, can be used as an internal standard. One
example is the use of stable isotopically labeled standards,
which are chemically identical to reagents used in the screen,
such as deuterium-labeled products. If these can be obtained,
they can have positive effects on the quality of a screen output
and so should be investigated early during assay development.
The use of an internal standard which can be added to the acid
in the stop step can also be considered. As seen in Table 2, a
product to standard ratio can be calculated, which mitigates for
the intra-well variation of product formation. This results in
lower standard deviations and % coefficient of variance of both
the vehicle and inhibitor controls.
13. The communication between the MS system and the acoustic
mist system is such that each scan performed on the MS is
annotated with the acoustic mist well location. This embedding of location within the raw data of the MS allows a single,
combined spectrum for each well on a plate to be produced in
an automated postprocessing algorithm (prototype in house
software) using the raw data. This embedding of well location
in the raw data also enables use of commercial applications such
as Genedata Expressionist and MassLynx for data parsing. A
full spectrum is produced for each well and is queried to
Mass Spectrometry for High-Throughput Screening
229
for the read. Using a low-volume reaction mixture and having a
large dilution to 50 μL final volume is advantageous when
using salts, as discussed in Note 6. Low-volume 384-well
polypropylene plates are a viable alternative, which would
allow lower volumes of reagents to be used. Currently, these
plates are expensive, so they may only be beneficial if the cost of
reagents outweighs the extra costs of the plates.
10. When storing assay plates to be read or re-read after more than
a day, they should be stored at ambient temperature rather than
cold. Storing assay plates at 4
C appears to reduce the ability to
fire on the MS. If plates are stored cold, they must be allowed
to fully equilibrate to ambient temperature prior to reading.
Plates should be stored in a stack or with a lid on to reduce
evaporation. Water may be added to the plates if the volume
has been reduced by evaporation when in storage.
11. It was decided to mask any well where the measured substrate
value is 0, but other low reading wells are taken through to
confirmation or IC/EC 50 screening to ensure that potential
hits are not missed.
12. Use of an internal standard in any AMI-MS assay greatly
improves ease of analysis as data can be referenced to and
normalized against this standard. Anything that does not interfere with the assay, such as a modified substrate or product or
any derivative of these, can be used as an internal standard. One
example is the use of stable isotopically labeled standards,
which are chemically identical to reagents used in the screen,
such as deuterium-labeled products. If these can be obtained,
they can have positive effects on the quality of a screen output
and so should be investigated early during assay development.
The use of an internal standard which can be added to the acid
in the stop step can also be considered. As seen in Table 2, a
product to standard ratio can be calculated, which mitigates for
the intra-well variation of product formation. This results in
lower standard deviations and % coefficient of variance of both
the vehicle and inhibitor controls.
13. The communication between the MS system and the acoustic
mist system is such that each scan performed on the MS is
annotated with the acoustic mist well location. This embedding of location within the raw data of the MS allows a single,
combined spectrum for each well on a plate to be produced in
an automated postprocessing algorithm (prototype in house
software) using the raw data. This embedding of well location
in the raw data also enables use of commercial applications such
as Genedata Expressionist and MassLynx for data parsing. A
full spectrum is produced for each well and is queried to
Mass Spectrometry for High-Throughput Screening
229