4 Notes
1. The optimum ammonium acetate concentration is proteindependent and can be optimized by testing concentrations
ranging from 10 mM up to 1 M. If needed, volatile additives
can be added, such as dithiothreitol or tris(2-carboxyethyl)
phosphine for disulfide bond reduction, imidazole, some detergents (see Note 10), or metallic ions in a volatile buffer (calcium acetate, zinc acetate, etc.).
2. Volatility of the buffer is key for native MS. Beware to avoid any
trace of nonvolatile salts such as sodium chloride. Ammonium
acetate resuspended at 200 mM in water is usually at pH 6.9. If
you want to adjust pH below 6.9, do not use HCl but formic
acid or acetic acid instead. Alternatively, you can dilute a commercial 7.5 M ammonium acetate solution (Sigma). Conversely, to increase the pH, use liquid NH 3 and not NaOH.
3. For smaller volumes, 7 K MWCO Zeba™ Micro Spin columns
(2–12 μL, Thermo Fisher Scientific) can be used.
4. Do not forget to turn off the cooling of the plate after use of
the nanomate. Otherwise, condensation will occur, water will
fill the wells, and the plate will have to be discarded the
next day.
5. The gas pressure and voltage to apply are the main two parameters to adjust. The stability of the spray can be visualized in
the Spray Optimization window, by pressing “Graph.” If the
intensity of the electrospray is dropping, incrementally increasing the gas pressure (no more than 0.5 psi) or the Voltage to
apply (no more than 1.80 kV) might help. If the spray is not
coming back, use another nozzle by pressing “Next Nozzle.”
Reducing the gas pressure and Voltage to apply while spraying
usually results in spray loss. The optimum Voltage to apply is
protein-dependent and has to be carefully optimized, as higher
values usually increase the ion transmission (more intense signal) and buffer removal (better resolved signal) but might
result in complex dissociation. Ticking “Return unused sample
to tray” enables to recover most of the sample after data
acquisition.
6. The backing pressure is a key parameter on Waters Instruments
(LCT, Synapt), which needs to be optimized for each protein
complex. Generally, this value is set around 6 mbar and can be
adjusted if necessary. This is possible by adding a homemade
system with two external valves that reduce the suction of the
primary pump.
188
Ste ´ phane Erb et al.
1. The optimum ammonium acetate concentration is proteindependent and can be optimized by testing concentrations
ranging from 10 mM up to 1 M. If needed, volatile additives
can be added, such as dithiothreitol or tris(2-carboxyethyl)
phosphine for disulfide bond reduction, imidazole, some detergents (see Note 10), or metallic ions in a volatile buffer (calcium acetate, zinc acetate, etc.).
2. Volatility of the buffer is key for native MS. Beware to avoid any
trace of nonvolatile salts such as sodium chloride. Ammonium
acetate resuspended at 200 mM in water is usually at pH 6.9. If
you want to adjust pH below 6.9, do not use HCl but formic
acid or acetic acid instead. Alternatively, you can dilute a commercial 7.5 M ammonium acetate solution (Sigma). Conversely, to increase the pH, use liquid NH 3 and not NaOH.
3. For smaller volumes, 7 K MWCO Zeba™ Micro Spin columns
(2–12 μL, Thermo Fisher Scientific) can be used.
4. Do not forget to turn off the cooling of the plate after use of
the nanomate. Otherwise, condensation will occur, water will
fill the wells, and the plate will have to be discarded the
next day.
5. The gas pressure and voltage to apply are the main two parameters to adjust. The stability of the spray can be visualized in
the Spray Optimization window, by pressing “Graph.” If the
intensity of the electrospray is dropping, incrementally increasing the gas pressure (no more than 0.5 psi) or the Voltage to
apply (no more than 1.80 kV) might help. If the spray is not
coming back, use another nozzle by pressing “Next Nozzle.”
Reducing the gas pressure and Voltage to apply while spraying
usually results in spray loss. The optimum Voltage to apply is
protein-dependent and has to be carefully optimized, as higher
values usually increase the ion transmission (more intense signal) and buffer removal (better resolved signal) but might
result in complex dissociation. Ticking “Return unused sample
to tray” enables to recover most of the sample after data
acquisition.
6. The backing pressure is a key parameter on Waters Instruments
(LCT, Synapt), which needs to be optimized for each protein
complex. Generally, this value is set around 6 mbar and can be
adjusted if necessary. This is possible by adding a homemade
system with two external valves that reduce the suction of the
primary pump.
188
Ste ´ phane Erb et al.