to perform the fractionation through five discontinuous steps
of acetonitrile (11.4%, 14.7%, 17.4%, 20.7%, and 50.0%) of
10 min each at a flow rate of 2 μL/min.
9. Adjust the second-dimension separation using an ACQUITY
UPLC M-Class HSS T3 Column using an acetonitrile gradient
from 7% to 40% (v/v) over 54 min at a flow rate of 0.4 μL/min
directly into a Synapt G2-Si HDMS.
10. Load 1 μL of 1 μg/μL samples into an ACQUITY UPLC
M-Class 2D System coupled to Synapt G2-Si HDMS and
perform HDMS
E acquisitions (see Note 11).
11. Access each chromatogram and, using the “Integrate Peaks”
tool, determine the relative proportion of sample volume for
the next steps (see Note 12).
3.5 Selection Rules
and UDMS
E Acquisition
1. Load Drift Scope software.
2. Using a selection tool, trace a line over the [M + 2H]
2+ charge
region (line A of Fig. 2) and then trace a line under the
[M + 2H]
2+ charged region (line B of Fig. 2) (see Note 13).
3. Export the selection rule as .rul file without cropping the data
(see Note 14).
4. In the MassLynx window, edit the existing HDMS
E method
correspondent to the first fraction (see Note 15).
5. Double click on the yellow bar.
6. Open the mobility tab, check the “Apply Rule file for Change
State/Drift Time Shipping” box, and open the selection rule
file previously saved.
7. Repeat those steps for the other fractions for each sample. This
step is time-consuming but ensures the best quality data.
8. After establishing the method for each fraction of each sample,
re-run the samples using their new, quasi-specific collision energies drift times-method and loading their relative volume previously calculated during Subheading 3.4, step 11.
3.6 Data Processing,
Quantification,
and Identification
1. Load Progenesis
® QI for Proteomics (version 4.0+) software to
process LC-MS/MS information (raw data).
2. Select and import the raw data for the first fraction, and provide
a lock mass of m/z 785.84261 to perform the calibration.
Select an appropriate *.fasta databank for threshold calculation.
3. After all files of the same fraction have been imported, click on
“Start Automatic Processing” and select to assess all runs in the
experiment for alignment suitability.
4. Check the box “Yes, automatically align my runs” and click”
Next.”
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Licia C. Silva-Costa et al.
of acetonitrile (11.4%, 14.7%, 17.4%, 20.7%, and 50.0%) of
10 min each at a flow rate of 2 μL/min.
9. Adjust the second-dimension separation using an ACQUITY
UPLC M-Class HSS T3 Column using an acetonitrile gradient
from 7% to 40% (v/v) over 54 min at a flow rate of 0.4 μL/min
directly into a Synapt G2-Si HDMS.
10. Load 1 μL of 1 μg/μL samples into an ACQUITY UPLC
M-Class 2D System coupled to Synapt G2-Si HDMS and
perform HDMS
E acquisitions (see Note 11).
11. Access each chromatogram and, using the “Integrate Peaks”
tool, determine the relative proportion of sample volume for
the next steps (see Note 12).
3.5 Selection Rules
and UDMS
E Acquisition
1. Load Drift Scope software.
2. Using a selection tool, trace a line over the [M + 2H]
2+ charge
region (line A of Fig. 2) and then trace a line under the
[M + 2H]
2+ charged region (line B of Fig. 2) (see Note 13).
3. Export the selection rule as .rul file without cropping the data
(see Note 14).
4. In the MassLynx window, edit the existing HDMS
E method
correspondent to the first fraction (see Note 15).
5. Double click on the yellow bar.
6. Open the mobility tab, check the “Apply Rule file for Change
State/Drift Time Shipping” box, and open the selection rule
file previously saved.
7. Repeat those steps for the other fractions for each sample. This
step is time-consuming but ensures the best quality data.
8. After establishing the method for each fraction of each sample,
re-run the samples using their new, quasi-specific collision energies drift times-method and loading their relative volume previously calculated during Subheading 3.4, step 11.
3.6 Data Processing,
Quantification,
and Identification
1. Load Progenesis
® QI for Proteomics (version 4.0+) software to
process LC-MS/MS information (raw data).
2. Select and import the raw data for the first fraction, and provide
a lock mass of m/z 785.84261 to perform the calibration.
Select an appropriate *.fasta databank for threshold calculation.
3. After all files of the same fraction have been imported, click on
“Start Automatic Processing” and select to assess all runs in the
experiment for alignment suitability.
4. Check the box “Yes, automatically align my runs” and click”
Next.”
160
Licia C. Silva-Costa et al.