these screens include Tris–HCl, tricine, and ammonium phosphate, at a maximum concentration of 20 mM.
2. Surfactants: These can both positively and negatively influence
detection of the substrate and product in the assay (see Note 3).
Detergents such as triton, DDM, CHAPS, and BSA have been
successful in AMI-MS screens.
3. Cleaning solution: 100% (v/v) ethanol.
2.6 Software
1. Microsoft
®
Excel, or other spreadsheet-based analysis
packages.
2. Genedata Screener
® (Genedata), or similar high-throughput
data analysis software.
3 Method
3.1 Instrument Setup
The schematic diagram (Fig. 2a) shows the principal components
involved in the mist ejection and the charging and ionization
processes.
3.1.1 Spray
Using an acoustic transducer, 20–80 nL aliquots of reaction mix are
fired, creating a chaotic mist droplet distribution that is sprayed
directly into the mass spectrometer (see Note 4). A custom csv file is
used to control the transducer.
3.1.2 Charging
The charging voltage will typically be +2.5 to 3 kV. This is optimum
for its two functions: to charge the liquid surface and to entrain and
focus the charge droplets leaving the surface of the liquid.
3.1.3 Polarity Switching
When charging the well to fire positively charged droplets, the well
becomes increasingly negatively charged. This prevents charge separation in the well and so prevents more positive droplets from
being fired. Polarity switching is therefore needed to return the
liquid in the well back to a neutral state to enable firing of further
positively charged droplets.
3.1.4 Target
Enhancement
This needs to be optimized to each particular screen around a set
mass that is appropriate for both product and substrate in the
reaction (Fig. 2b) (see Note 5). This generally increases sensitivity
around four- to fivefold versus a non-enhanced acquisition. The
user needs to be aware that masses outside or at the edge of the
enhancement window will be understated or undetectable.
3.1.5 Buffer
Considerations for Acoustic
Settings
For simple aqueous buffers, default screening run protocols from
the acoustic mist system will be applicable. The default settings will
have typical parameters of a desolvation temperature (transfer tube) of
300–330
C and a cone gas flow that is between 30 and 80 L/h
220
Helen Plant et al.
2. Surfactants: These can both positively and negatively influence
detection of the substrate and product in the assay (see Note 3).
Detergents such as triton, DDM, CHAPS, and BSA have been
successful in AMI-MS screens.
3. Cleaning solution: 100% (v/v) ethanol.
2.6 Software
1. Microsoft
®
Excel, or other spreadsheet-based analysis
packages.
2. Genedata Screener
® (Genedata), or similar high-throughput
data analysis software.
3 Method
3.1 Instrument Setup
The schematic diagram (Fig. 2a) shows the principal components
involved in the mist ejection and the charging and ionization
processes.
3.1.1 Spray
Using an acoustic transducer, 20–80 nL aliquots of reaction mix are
fired, creating a chaotic mist droplet distribution that is sprayed
directly into the mass spectrometer (see Note 4). A custom csv file is
used to control the transducer.
3.1.2 Charging
The charging voltage will typically be +2.5 to 3 kV. This is optimum
for its two functions: to charge the liquid surface and to entrain and
focus the charge droplets leaving the surface of the liquid.
3.1.3 Polarity Switching
When charging the well to fire positively charged droplets, the well
becomes increasingly negatively charged. This prevents charge separation in the well and so prevents more positive droplets from
being fired. Polarity switching is therefore needed to return the
liquid in the well back to a neutral state to enable firing of further
positively charged droplets.
3.1.4 Target
Enhancement
This needs to be optimized to each particular screen around a set
mass that is appropriate for both product and substrate in the
reaction (Fig. 2b) (see Note 5). This generally increases sensitivity
around four- to fivefold versus a non-enhanced acquisition. The
user needs to be aware that masses outside or at the edge of the
enhancement window will be understated or undetectable.
3.1.5 Buffer
Considerations for Acoustic
Settings
For simple aqueous buffers, default screening run protocols from
the acoustic mist system will be applicable. The default settings will
have typical parameters of a desolvation temperature (transfer tube) of
300–330
C and a cone gas flow that is between 30 and 80 L/h
220
Helen Plant et al.