7. Place the column in a 1-mL Eppendorf tube and elute the
desalted proteins by centrifuging for 4 min at 1,000 Â g
(Micro Bio-Spin™) or 2 min at 1,500 Â g (Zeba™).
3.2 Ultrafiltration
Desalting
1. Operate at +4
C.
2. After selecting the appropriate MWCO of the ultrafiltration
device, equilibrate its membrane with 500 μL of 200 mM
ammonium acetate. Typical devices include Amicon™ Ultra
0.5 mL or Vivaspin™ 500 centrifugal concentrators (Merck).
Centrifuge at 10,000 Â g for 30 s, discard the eluate, and repeat
three times.
3. Load your sample (up to 500 μL).
4. Concentrate down to 50 μL at 10,000 Â g. Make up to 500 μL
with 200 mM ammonium acetate and repeat five times this
dilution/concentration cycle.
3.3 Direct Infusion of
the Sample with the
TriVersa Nanomate™
(Advion)
1. In the interface setting panel, set the controller power On and
the temperature to 4
C (see Note 4).
2. Load 10 μL of desalted protein sample to the 96- or 384-well
plate.
3. In the Spray Optimization panel, set the Sample Volume to
5 μL, the gas pressure to 0.35 psi, the Voltage to apply to
1.60 kV and press “Deliver Sample” (see Note 5).
3.4 Calibration
3.4.1 TOF LCT (Upgraded
for High Mass Detection by
MS Vision)
1. Using the TriVersa Nanomate™ (see Subheading 3.3), deliver
5 μL of cesium iodide calibration mix (see Subheading 2.2).
2. In MassLynx MS Tune window, manually set the following
parameters: Operate the instrument in positive mode with an
m/z range from 1,000 to 10,000. Apply 120 V and 5 V for the
sampling cone and extraction cone, respectively. The source
temperature is set at 90
C, but the capillary voltage and the gas
flows are not used in the TriVersa Nanomate™ interface setup.
3. Adjust the backing pressure to 6 mbar (see Note 6).
4. Start a 2-min acquisition with 4-s scans.
5. Average the signal over the 2 min in the Chromatogram panel
and in the Spectrum panel, select “Tools,” then “Make Calibration,” and select “CsI_esi.ref” profile. Adjust calibration
points for the m/z range targeted and click on File\Save Spectrum, then click OK and finally accept the calibration after
closing the window.
6. Do not forget to load this calibration file (e.g., named CsI_date) before starting the first acquisition of the analyzed sample. To do so, click on “Acquire,” then select “calibration,” and
load the calibration file. Finally perform the acquisition.
176
Ste ´ phane Erb et al.
desalted proteins by centrifuging for 4 min at 1,000 Â g
(Micro Bio-Spin™) or 2 min at 1,500 Â g (Zeba™).
3.2 Ultrafiltration
Desalting
1. Operate at +4
C.
2. After selecting the appropriate MWCO of the ultrafiltration
device, equilibrate its membrane with 500 μL of 200 mM
ammonium acetate. Typical devices include Amicon™ Ultra
0.5 mL or Vivaspin™ 500 centrifugal concentrators (Merck).
Centrifuge at 10,000 Â g for 30 s, discard the eluate, and repeat
three times.
3. Load your sample (up to 500 μL).
4. Concentrate down to 50 μL at 10,000 Â g. Make up to 500 μL
with 200 mM ammonium acetate and repeat five times this
dilution/concentration cycle.
3.3 Direct Infusion of
the Sample with the
TriVersa Nanomate™
(Advion)
1. In the interface setting panel, set the controller power On and
the temperature to 4
C (see Note 4).
2. Load 10 μL of desalted protein sample to the 96- or 384-well
plate.
3. In the Spray Optimization panel, set the Sample Volume to
5 μL, the gas pressure to 0.35 psi, the Voltage to apply to
1.60 kV and press “Deliver Sample” (see Note 5).
3.4 Calibration
3.4.1 TOF LCT (Upgraded
for High Mass Detection by
MS Vision)
1. Using the TriVersa Nanomate™ (see Subheading 3.3), deliver
5 μL of cesium iodide calibration mix (see Subheading 2.2).
2. In MassLynx MS Tune window, manually set the following
parameters: Operate the instrument in positive mode with an
m/z range from 1,000 to 10,000. Apply 120 V and 5 V for the
sampling cone and extraction cone, respectively. The source
temperature is set at 90
C, but the capillary voltage and the gas
flows are not used in the TriVersa Nanomate™ interface setup.
3. Adjust the backing pressure to 6 mbar (see Note 6).
4. Start a 2-min acquisition with 4-s scans.
5. Average the signal over the 2 min in the Chromatogram panel
and in the Spectrum panel, select “Tools,” then “Make Calibration,” and select “CsI_esi.ref” profile. Adjust calibration
points for the m/z range targeted and click on File\Save Spectrum, then click OK and finally accept the calibration after
closing the window.
6. Do not forget to load this calibration file (e.g., named CsI_date) before starting the first acquisition of the analyzed sample. To do so, click on “Acquire,” then select “calibration,” and
load the calibration file. Finally perform the acquisition.
176
Ste ´ phane Erb et al.