7. Reference files for higher m/z ranges can be custom made. In
the case of CsI, increments of +259.81 Da can be directly
added to the initial CsI calibration file.
8. In order to optimally accumulate and transfer heavy ions,
transfer time and prepulse storage time parameters should be
increased compared to small ions analysis. These two parameters are linked, but the transfer time has to be higher than
the prepusle storage time.
9. Waters TOF or Q-TOF mass spectrometers can be upgraded
(MS Vision, the Netherlands) by modifying electronics and gas
pressure in order to improve the transmission of high molecular mass species in non-denaturing conditions. On our
upgraded LCT, the combination of an additional gas arrival
(argon) in the first hexapole housing through a needle valve
and the modification of different electronic parts (e.g., higher
voltages applied to sample and extraction cones) results in an
improvement of high molecular mass transmission. Typically,
the sensitivity for GroEL14 analysis is increased sixfold by
argon admission if the pressure is set around 4 Â 10
À2 mbar.
A micro-Pirani gauge is installed to control the pressure
applied.
10. n-Dodecyl β-D-maltoside can be used at two times the critical
micellar concentration (0.02%) in order to stabilize very labile
soluble complexes. It can also be used at the same concentration to spray membrane proteins [29, 30].
11. The m/z range on Waters Q-TOF depends on the type of
Quadrupole that is installed: up to 16,000 m/z for a
4,000 m/z quadrupole, up to 32,000 m/z for an 8,000 m/z
quadrupole and up to 128,000 m/z for a 32,000 m/z
quadrupole.
12. The sample cone is a key parameter on Waters Instruments
(LCT, Synapt, etc.) that needs to be optimized for each protein
complex. Similarly, to the voltage applied to the nozzle of the
TriVersa Nanomate™, higher values usually increase the intensity of the signal and provide better resolved peaks by removing
residual buffer. However, high sample cone values might result
in complex dissociation. Here again, an optimum value has to
be found that provides good signal without undermining the
multiprotein complex integrity. A good strategy is to start with
a maximum Sampling Cone voltage (150 V) and progressively
reduce it by 10-V steps.
13. The trap collision energy is another key parameter on Waters
Q-TOF. In the case of membrane proteins or very large multiprotein complexes (>500 kDa), increasing the trap collision
energy might be helpful or even mandatory in order to thermodynamically cool down the ions (large complexes) or
Native Mass Spectrometry
189
the case of CsI, increments of +259.81 Da can be directly
added to the initial CsI calibration file.
8. In order to optimally accumulate and transfer heavy ions,
transfer time and prepulse storage time parameters should be
increased compared to small ions analysis. These two parameters are linked, but the transfer time has to be higher than
the prepusle storage time.
9. Waters TOF or Q-TOF mass spectrometers can be upgraded
(MS Vision, the Netherlands) by modifying electronics and gas
pressure in order to improve the transmission of high molecular mass species in non-denaturing conditions. On our
upgraded LCT, the combination of an additional gas arrival
(argon) in the first hexapole housing through a needle valve
and the modification of different electronic parts (e.g., higher
voltages applied to sample and extraction cones) results in an
improvement of high molecular mass transmission. Typically,
the sensitivity for GroEL14 analysis is increased sixfold by
argon admission if the pressure is set around 4 Â 10
À2 mbar.
A micro-Pirani gauge is installed to control the pressure
applied.
10. n-Dodecyl β-D-maltoside can be used at two times the critical
micellar concentration (0.02%) in order to stabilize very labile
soluble complexes. It can also be used at the same concentration to spray membrane proteins [29, 30].
11. The m/z range on Waters Q-TOF depends on the type of
Quadrupole that is installed: up to 16,000 m/z for a
4,000 m/z quadrupole, up to 32,000 m/z for an 8,000 m/z
quadrupole and up to 128,000 m/z for a 32,000 m/z
quadrupole.
12. The sample cone is a key parameter on Waters Instruments
(LCT, Synapt, etc.) that needs to be optimized for each protein
complex. Similarly, to the voltage applied to the nozzle of the
TriVersa Nanomate™, higher values usually increase the intensity of the signal and provide better resolved peaks by removing
residual buffer. However, high sample cone values might result
in complex dissociation. Here again, an optimum value has to
be found that provides good signal without undermining the
multiprotein complex integrity. A good strategy is to start with
a maximum Sampling Cone voltage (150 V) and progressively
reduce it by 10-V steps.
13. The trap collision energy is another key parameter on Waters
Q-TOF. In the case of membrane proteins or very large multiprotein complexes (>500 kDa), increasing the trap collision
energy might be helpful or even mandatory in order to thermodynamically cool down the ions (large complexes) or
Native Mass Spectrometry
189