3.6.3 DDA (IDA) Analysis
of Sample Proteolytic
Digests to Build a Spectral
Ion Library
The pooled sample from each group is analyzed in the IDA mode to
generate the spectral library.
Create a DDA/IDA method using Analyst TF 1.7.1 with one
survey scan and 60 candidate ion scans per cycle (see Note 8).
TOF MS (MS1)
1. Be sure that the duration of the method is the same as that of
the LC gradient method, and select the accumulation time to
250 ms and the mass range from 350 to 1400 Da (see Note 9).
2. In the Switch Criteria tab set, the range to match that which
you selected under the above window, monitor charge states
from 2 to 5, which exceed 150 counts, and set the mass
tolerance to 100 mDa.
3. In the Include/Exclude or in the IDA Advanced tab, make
any masses you want to monitor or exclude the selection of the
collision energy or any changes that would be necessary for
your precise experiment. In this case, the default settings do
not need to be changed.
Product Ion (MS2)
1. In the MS Tab, set the accumulation time to 100 ms and the
mass range from 100 to 1500 Da and select high resolution or
high sensitivity in the function of your assay (for proteomics
assays, the most common is selecting high sensitivity).
2. All other tabs do not need to be changed because the parameters are the same as for the TOF MS.
3. Then, select the Gradient accord with your sample type (in our
case, we perform a 40-min gradient in micro mode (5 μL/
min)), select the Loading Pump (in our case, load during 3 min
10 μL/min) and autosampler methods (in our case, we load
4 μL from a 1-mg/mL peptide solution) and then save your
Acquisition File.
4. Run your samples.
SWATH-MS Data
Acquisition (DIA-MS
Acquisition)
SWATH-MS workflows can be designed in two different fashions,
either using a fixed size of transmission window (FW) or using
variable window size (VW). The aim of designing a VW method is
to increase the selectivity of the experiment while maintaining the
sensitivity. Whereas we can create an FW experiment before we
know the complexity of the sample of interest, in the case of VW
design, we need to have the full chromatographic MS data. This
information can be found analyzing the whole MS experiment of
one of the library runs. The extraction of all the precursor information from these runs using software called PeakView
® and generates
the entries needed for the Variable Window Calculator. With this
tool, all the Q1 transmission windows needed are generated. The
windows generated are exported to a text file that can finally be
integrated into the Acquisition Control software. Other parameters
132
Maria del Pilar Chantada-Va ´ zquez et al.
of Sample Proteolytic
Digests to Build a Spectral
Ion Library
The pooled sample from each group is analyzed in the IDA mode to
generate the spectral library.
Create a DDA/IDA method using Analyst TF 1.7.1 with one
survey scan and 60 candidate ion scans per cycle (see Note 8).
TOF MS (MS1)
1. Be sure that the duration of the method is the same as that of
the LC gradient method, and select the accumulation time to
250 ms and the mass range from 350 to 1400 Da (see Note 9).
2. In the Switch Criteria tab set, the range to match that which
you selected under the above window, monitor charge states
from 2 to 5, which exceed 150 counts, and set the mass
tolerance to 100 mDa.
3. In the Include/Exclude or in the IDA Advanced tab, make
any masses you want to monitor or exclude the selection of the
collision energy or any changes that would be necessary for
your precise experiment. In this case, the default settings do
not need to be changed.
Product Ion (MS2)
1. In the MS Tab, set the accumulation time to 100 ms and the
mass range from 100 to 1500 Da and select high resolution or
high sensitivity in the function of your assay (for proteomics
assays, the most common is selecting high sensitivity).
2. All other tabs do not need to be changed because the parameters are the same as for the TOF MS.
3. Then, select the Gradient accord with your sample type (in our
case, we perform a 40-min gradient in micro mode (5 μL/
min)), select the Loading Pump (in our case, load during 3 min
10 μL/min) and autosampler methods (in our case, we load
4 μL from a 1-mg/mL peptide solution) and then save your
Acquisition File.
4. Run your samples.
SWATH-MS Data
Acquisition (DIA-MS
Acquisition)
SWATH-MS workflows can be designed in two different fashions,
either using a fixed size of transmission window (FW) or using
variable window size (VW). The aim of designing a VW method is
to increase the selectivity of the experiment while maintaining the
sensitivity. Whereas we can create an FW experiment before we
know the complexity of the sample of interest, in the case of VW
design, we need to have the full chromatographic MS data. This
information can be found analyzing the whole MS experiment of
one of the library runs. The extraction of all the precursor information from these runs using software called PeakView
® and generates
the entries needed for the Variable Window Calculator. With this
tool, all the Q1 transmission windows needed are generated. The
windows generated are exported to a text file that can finally be
integrated into the Acquisition Control software. Other parameters
132
Maria del Pilar Chantada-Va ´ zquez et al.