the adequate search method; select tryptic digestion Carbamidomethylation as a fixed modification and Methionine oxidation as a variable modification.
3. Once the search is completed in the “FDR report” generated,
there is an important number that is the proteins identified
with 1% Global FDR, which is necessary to be used as input to
import the ion library using the SWATH-MS MicroApp.
Importing Ion Libraries into
the SWATH-MS Microapp
and Analyzing SWATHMS Data
1. Open PeakView, and at the top of the screen, select Quantization ! SWATH-MS Processing ! Import Ion Library.
2. Find the .group file produced from the Protein Pilot search and
set the number of proteins (selected in the previous section) to
import to the 1% Global FDR (see Note 11); usually, the
peptides shared by more than one protein are not imported.
Under Select sample type, choose the option appropriate for
whether the samples are unlabeled (typical) or labeled with a
chemical tag (i.e., iTRAQ, SILAC).
3. Select all of the SWATH-MS files to be analyzed for a given
experiment.
4. Set your processing settings. For protein quantization analysis,
typical parameter settings are discussed in Note 10.
5. Perform the RT calibration using peptides from your own
proteins. Once the calibration curve is good, click apply, and
all the RT will be calibrated.
6. Click “Process” to analyze your SWATH-MS data.
7. Once completed, you can export the data for the analysis in
MarkerView by clicking Quantization ! SWATH-MS Processing ! Export ! Areas or Export ! All to get a complete
list of all parameters for the analysis in Excel format.
4 Notes
1. All chemicals used for the preparation of LC solvents should be
LC-MS grade in order to avoid possible particles that can
contaminate the machine or give erroneous protein
identification.
2. There are a lot of protease inhibitor kits that prevent protein
degradation. Each kit of proteases has little differences in their
composition. You have to select the most adequate to your
sample.
3. Gels were prepared using tables found in E. Harlow and D.
Lane (Editors), Antibodies: A Laboratory Manual. XIII + 726
S., 50 Abb., 62 Tab. Cold Spring Harbor 1988. ISBN: 087969-314–2.
134
Maria del Pilar Chantada-Va ´ zquez et al.
3. Once the search is completed in the “FDR report” generated,
there is an important number that is the proteins identified
with 1% Global FDR, which is necessary to be used as input to
import the ion library using the SWATH-MS MicroApp.
Importing Ion Libraries into
the SWATH-MS Microapp
and Analyzing SWATHMS Data
1. Open PeakView, and at the top of the screen, select Quantization ! SWATH-MS Processing ! Import Ion Library.
2. Find the .group file produced from the Protein Pilot search and
set the number of proteins (selected in the previous section) to
import to the 1% Global FDR (see Note 11); usually, the
peptides shared by more than one protein are not imported.
Under Select sample type, choose the option appropriate for
whether the samples are unlabeled (typical) or labeled with a
chemical tag (i.e., iTRAQ, SILAC).
3. Select all of the SWATH-MS files to be analyzed for a given
experiment.
4. Set your processing settings. For protein quantization analysis,
typical parameter settings are discussed in Note 10.
5. Perform the RT calibration using peptides from your own
proteins. Once the calibration curve is good, click apply, and
all the RT will be calibrated.
6. Click “Process” to analyze your SWATH-MS data.
7. Once completed, you can export the data for the analysis in
MarkerView by clicking Quantization ! SWATH-MS Processing ! Export ! Areas or Export ! All to get a complete
list of all parameters for the analysis in Excel format.
4 Notes
1. All chemicals used for the preparation of LC solvents should be
LC-MS grade in order to avoid possible particles that can
contaminate the machine or give erroneous protein
identification.
2. There are a lot of protease inhibitor kits that prevent protein
degradation. Each kit of proteases has little differences in their
composition. You have to select the most adequate to your
sample.
3. Gels were prepared using tables found in E. Harlow and D.
Lane (Editors), Antibodies: A Laboratory Manual. XIII + 726
S., 50 Abb., 62 Tab. Cold Spring Harbor 1988. ISBN: 087969-314–2.
134
Maria del Pilar Chantada-Va ´ zquez et al.