4. Add 25 μL of 0.2% RapiGest SF and mix well.
5. Heat microtubes using a block heater or thermoshaker at 80
C
for 15 min.
6. Remove the tubes from the heater or thermoshaker and centrifuge briefly to remove any condensation on the tube walls and
lid. Set the temperature to 60
C.
7. Add 2.5 μL of dithiothreitol solution and mix.
8. Heat microtubes using a block heater or thermoshaker at 60
C
for 30 min.
9. Remove the samples from the block heater or thermoshaker
and allow them to cool to room temperature.
10. Add 2.5 μL of iodoacetamide solution and vortex to mix.
11. Incubate the samples for 30 min at room temperature in
the dark.
12. Add 10 μL of trypsin solution and vortex (see Note 9).
13. Incubate the samples at 37
C overnight (see Note 10).
14. Add 10 μL of 5% TFA to hydrolyze the RapiGest and mix by
inversion.
15. Incubate the samples at 37
C for 90 min.
16. Centrifuge the samples at 20,800 Â g and 6
C for 30 min.
17. Transfer the supernatants to Waters Total Recovery vials.
18. Add a proportional amount of enolase so that each injected
fraction has 0.5 μg of protein digest and 80 fmol/μL of
enolase.
3.4 HDMS
E Spectra
Acquisition
and Scouting Runs
1. Establish solutions C and D as mobile phases of first-dimension
chromatography.
2. Set up solutions E and F as mobile phases of second-dimension
chromatography.
3. Calibrate the mass spectrometer according to the manufacturer’s instructions.
4. Set the MS acquisition method to alternate between low and
high energy, with no selection window and a continuous ion
current.
5. Use a CE ramp from 19 eV to 53 eV in the transfer cell for the
elevated energy MS scan.
6. Set Nanoflow conditions on the Tune Page.
7. Set the mass spectrometer to operate in resolution mode with
an m/z ratio resolving power of at least 40,000 FWHM, using
ion mobility with a cross-section resolving power of 40 Ω/ΔΩ.
8. Adjust the first-dimension chromatography method on an
ACQUITY UPLC M-Class Peptide BEH C18 Trap Column
Human Blood Plasma Investigation Employing 2D UPLC-UDMS
E Data. . .
159
5. Heat microtubes using a block heater or thermoshaker at 80
C
for 15 min.
6. Remove the tubes from the heater or thermoshaker and centrifuge briefly to remove any condensation on the tube walls and
lid. Set the temperature to 60
C.
7. Add 2.5 μL of dithiothreitol solution and mix.
8. Heat microtubes using a block heater or thermoshaker at 60
C
for 30 min.
9. Remove the samples from the block heater or thermoshaker
and allow them to cool to room temperature.
10. Add 2.5 μL of iodoacetamide solution and vortex to mix.
11. Incubate the samples for 30 min at room temperature in
the dark.
12. Add 10 μL of trypsin solution and vortex (see Note 9).
13. Incubate the samples at 37
C overnight (see Note 10).
14. Add 10 μL of 5% TFA to hydrolyze the RapiGest and mix by
inversion.
15. Incubate the samples at 37
C for 90 min.
16. Centrifuge the samples at 20,800 Â g and 6
C for 30 min.
17. Transfer the supernatants to Waters Total Recovery vials.
18. Add a proportional amount of enolase so that each injected
fraction has 0.5 μg of protein digest and 80 fmol/μL of
enolase.
3.4 HDMS
E Spectra
Acquisition
and Scouting Runs
1. Establish solutions C and D as mobile phases of first-dimension
chromatography.
2. Set up solutions E and F as mobile phases of second-dimension
chromatography.
3. Calibrate the mass spectrometer according to the manufacturer’s instructions.
4. Set the MS acquisition method to alternate between low and
high energy, with no selection window and a continuous ion
current.
5. Use a CE ramp from 19 eV to 53 eV in the transfer cell for the
elevated energy MS scan.
6. Set Nanoflow conditions on the Tune Page.
7. Set the mass spectrometer to operate in resolution mode with
an m/z ratio resolving power of at least 40,000 FWHM, using
ion mobility with a cross-section resolving power of 40 Ω/ΔΩ.
8. Adjust the first-dimension chromatography method on an
ACQUITY UPLC M-Class Peptide BEH C18 Trap Column
Human Blood Plasma Investigation Employing 2D UPLC-UDMS
E Data. . .
159