either or both production and read processes as automated
procedures.
3.5 Instrument
Cleaning
and Maintenance
The main reason for cleaning is due to the constant flow of air
through the system, entering via the charging cone. The charging
cone therefore becomes contaminated, which can change the field,
leading to variability in sensitivity and edge effects on the plate.
Cleaning processes for the AMI-MS include removal of the charging cone and sonication in 100% (v/v) ethanol. This process is
recommended to be carried out on a weekly basis. The primary
maintenance required for the Echo
® is around the acoustic coupling fluidics. As this involves recirculated water, evaporation
occurs so that regular refilling is necessary. Also, as this is open to
the atmosphere, microbial contamination can be an issue. At each
water change (recommended weekly), the fluidics need to be backflushed and a fresh bottle of water fitted. Preventative maintenance
on all instruments as per the manufacturer’s schedule should also
be performed.
3.6 Data Acquisition
Plate reading in AMI-MS has a low failure rate; however, around
0.1% of wells do fail to fire. This can give a false hit in the analysis,
due to apparent inhibition, if the substrate appears to be low, or
zero, when the ratio is calculated (see Note 11).
Well-to-well variability can be seen with AMI-MS assays due to
the high sensitivity of the technology. In this way, a small change in
firing volume can have large consequences on assay output. Firing
of the droplets from the Echo
® system is variable, which means that
wells can appear to have different levels of substrate or product, due
to the variation in the amount of liquid fired. For this reason, it is
recommended where possible to use an internal standard in the
assay to allow normalization across wells and plates (Fig. 3) (see
Note 12).
The hit rate tends to be low for AMI-MS assays as there are few
artifacts compared to traditional technologies such as fluorescence
intensity where, for example, false positives may be identified in the
form of fluorescent compounds. Since AMI-MS only detects the
substrate and product of interest using mass detection, potential
artifacts may be limited to compounds that cause mass interference.
Mass interference is an issue caused by utilization of an internal
standard in the assay, although this does bring advantages in the
quality of the screening output (Fig. 3) (Table 2). Mass interference
occurs if a compound, a fragment of a compound, or a contaminant
of a compound being tested, is the same mass as the internal
standard. This leads to such a compound appearing active when
the ratio of product/internal standard is calculated, as the internal
standard layer will appear large and therefore the ratio will be small.
This manifests as a low product result (well appears inhibited) when
in reality the compound is simply interfering with the ratio
Mass Spectrometry for High-Throughput Screening
225
procedures.
3.5 Instrument
Cleaning
and Maintenance
The main reason for cleaning is due to the constant flow of air
through the system, entering via the charging cone. The charging
cone therefore becomes contaminated, which can change the field,
leading to variability in sensitivity and edge effects on the plate.
Cleaning processes for the AMI-MS include removal of the charging cone and sonication in 100% (v/v) ethanol. This process is
recommended to be carried out on a weekly basis. The primary
maintenance required for the Echo
® is around the acoustic coupling fluidics. As this involves recirculated water, evaporation
occurs so that regular refilling is necessary. Also, as this is open to
the atmosphere, microbial contamination can be an issue. At each
water change (recommended weekly), the fluidics need to be backflushed and a fresh bottle of water fitted. Preventative maintenance
on all instruments as per the manufacturer’s schedule should also
be performed.
3.6 Data Acquisition
Plate reading in AMI-MS has a low failure rate; however, around
0.1% of wells do fail to fire. This can give a false hit in the analysis,
due to apparent inhibition, if the substrate appears to be low, or
zero, when the ratio is calculated (see Note 11).
Well-to-well variability can be seen with AMI-MS assays due to
the high sensitivity of the technology. In this way, a small change in
firing volume can have large consequences on assay output. Firing
of the droplets from the Echo
® system is variable, which means that
wells can appear to have different levels of substrate or product, due
to the variation in the amount of liquid fired. For this reason, it is
recommended where possible to use an internal standard in the
assay to allow normalization across wells and plates (Fig. 3) (see
Note 12).
The hit rate tends to be low for AMI-MS assays as there are few
artifacts compared to traditional technologies such as fluorescence
intensity where, for example, false positives may be identified in the
form of fluorescent compounds. Since AMI-MS only detects the
substrate and product of interest using mass detection, potential
artifacts may be limited to compounds that cause mass interference.
Mass interference is an issue caused by utilization of an internal
standard in the assay, although this does bring advantages in the
quality of the screening output (Fig. 3) (Table 2). Mass interference
occurs if a compound, a fragment of a compound, or a contaminant
of a compound being tested, is the same mass as the internal
standard. This leads to such a compound appearing active when
the ratio of product/internal standard is calculated, as the internal
standard layer will appear large and therefore the ratio will be small.
This manifests as a low product result (well appears inhibited) when
in reality the compound is simply interfering with the ratio
Mass Spectrometry for High-Throughput Screening
225