source parameters, set the capillary voltage at 4 kV, the nebulizer at 0.4 bar, the dry gas at 4.0 L/min, and the temperature
at 220
C.
3. To perform the calibration, go to the “Calibration” tab and
select the CsI reference file, then check that the calibration
mode is on “Enhanced Quadratic Mode.” Finally, click to
“Calibrate” and accept the calibration.
3.4.4 Exactive™ Plus
EMR Orbitrap
1. Using the HESI source and the 500 μL Hamilton™ syringe,
deliver cesium iodide calibration mix (see Subheading 2.2) with
a flow rate of 10 μL/min.
2. In the MS Tune page, manually set the following parameters:
Operate the instrument in positive mode with an m/z
range from 1,000 to 20,000. Apply 25 eV and 100 eV for the
CID and CE parameters, respectively. The source temperature
is set at 250
C, the capillary voltage is set to 4 kV, and the gas
flow is set to 10 u.a. The trapping gas pressure is set to 7 u.a.
Set the ion optics (injection, inter, and bent flatapoles) at 4 V,
the nominal resolution at 17,500 and activate the EMR mode.
3. To perform the calibration, wait for a stable Total Ion Chromatogram (<12%), then select “Calibrate,” and check “EMR
MS Mass Calibration (pos)”.
3.4.5 Q-Exactive™
BioPharma Orbitrap
1. Using the syringe pusher to deliver the ammonium hexafluorophosphate calibration mix at 5 μL/min.
2. In the Calibration Panel, select “HMR Mode calibration
(pos).”.
3.5 Native MS
Analysis of E. coli RNA
Polymerase on a
Modified TOF LCT
High mass detection on a modified ESI-TOF is illustrated by the
analysis of E. coli RNA polymerase, a $400 kDa hexameric enzyme
consisting of a dimer of α subunits bound to single copies of β, β
0 ,
ω, and a σ-subunits [17]. Here, a complex purified using an established procedure [18] is characterized using a simple TOF analyzer,
previously optimized for the transfer of high-molecular weight
species (see Note 9).
1. Desalt approximatively 100 μL of E. coli RNA polymerase at
30 μM in 500 mM ammonium acetate at pH 6.9 as explained in
Subheading 3.2 using a Vivaspin™ 100 kDa cut-off membrane
at 15,000 Â g and +4
C.
2. Infuse the sample at 5 μM with the TriVersa Nanomate™ (see
Subheading 3.3).
3. Start with the same parameters as the ones used for CsI calibration but set the cone voltage to 150 V and the extraction cone
to 50 V. Increase the backing pressure up to 7 mbar (see Note
6). Adjust the pressure inside the first hexapole ion guide to
178
Ste ´ phane Erb et al.
at 220
C.
3. To perform the calibration, go to the “Calibration” tab and
select the CsI reference file, then check that the calibration
mode is on “Enhanced Quadratic Mode.” Finally, click to
“Calibrate” and accept the calibration.
3.4.4 Exactive™ Plus
EMR Orbitrap
1. Using the HESI source and the 500 μL Hamilton™ syringe,
deliver cesium iodide calibration mix (see Subheading 2.2) with
a flow rate of 10 μL/min.
2. In the MS Tune page, manually set the following parameters:
Operate the instrument in positive mode with an m/z
range from 1,000 to 20,000. Apply 25 eV and 100 eV for the
CID and CE parameters, respectively. The source temperature
is set at 250
C, the capillary voltage is set to 4 kV, and the gas
flow is set to 10 u.a. The trapping gas pressure is set to 7 u.a.
Set the ion optics (injection, inter, and bent flatapoles) at 4 V,
the nominal resolution at 17,500 and activate the EMR mode.
3. To perform the calibration, wait for a stable Total Ion Chromatogram (<12%), then select “Calibrate,” and check “EMR
MS Mass Calibration (pos)”.
3.4.5 Q-Exactive™
BioPharma Orbitrap
1. Using the syringe pusher to deliver the ammonium hexafluorophosphate calibration mix at 5 μL/min.
2. In the Calibration Panel, select “HMR Mode calibration
(pos).”.
3.5 Native MS
Analysis of E. coli RNA
Polymerase on a
Modified TOF LCT
High mass detection on a modified ESI-TOF is illustrated by the
analysis of E. coli RNA polymerase, a $400 kDa hexameric enzyme
consisting of a dimer of α subunits bound to single copies of β, β
0 ,
ω, and a σ-subunits [17]. Here, a complex purified using an established procedure [18] is characterized using a simple TOF analyzer,
previously optimized for the transfer of high-molecular weight
species (see Note 9).
1. Desalt approximatively 100 μL of E. coli RNA polymerase at
30 μM in 500 mM ammonium acetate at pH 6.9 as explained in
Subheading 3.2 using a Vivaspin™ 100 kDa cut-off membrane
at 15,000 Â g and +4
C.
2. Infuse the sample at 5 μM with the TriVersa Nanomate™ (see
Subheading 3.3).
3. Start with the same parameters as the ones used for CsI calibration but set the cone voltage to 150 V and the extraction cone
to 50 V. Increase the backing pressure up to 7 mbar (see Note
6). Adjust the pressure inside the first hexapole ion guide to
178
Ste ´ phane Erb et al.