8. On the final tab “Run,” set the output directory to an
appropriate location (e.g., C:\FragPipe\tutorial_output\), and
then click “RUN” near the top left. The entire analysis workflow will now commence. This will take about an hour
depending on the speed of your computer. After successful
processing, you should find several new files in the folder you
specified including “combined.pep.xml,” which we will use in
the next section.
3.2 Quantification
of Peptides
and Detection
of Protein Changes
1. The combined peptide identifications from Subheading 3.1 are
imported into the Skyline software for peptide-level quantification. Open Skyline and select “Import DDA Peptide Search”
from the options on the start page. You will be prompted to
save the document.
2. In the “Import Peptide Search” box that pops up, change the
cutoff score to “0.99,” click “Add Files. . .” on the right side,
and select the output from FragPipe in the last section “combined.pep.xml.” Click “Next” to start building the spectral
library. This step will take about 10 min with the tutorial
data, and the duration is proportional to the number of confident peptide-spectrum matches produced by FragPipe/MSFragger analysis (see Note 3).
3. Once the library is finished building, the box will change to
“Extract Chromatograms,” and Skyline should find your
source mzML files and populate the “Results files found” list.
If your files are not in the list, browse the file system and add
them. Click “Next.” A box should pop up asking if you want to
remove the common file prefix. This makes viewing the replicates easier. Click “OK” to remove the prefix.
4. The box will change to “Add Modifications” and will show a
list of modifications found in the library that you can choose
to include in the quantification. For the purpose of our
analysis, check the box next to “Acetyl (N-term) ¼ [42]”
and “Oxidation (M) ¼ M[16],” and then click “Next >” at
the bottom.
5. “Configure Full-Scan Settings” will appear. Set precursor
charges to “2, 3, 4, 5.” Under “Isotope peaks included,” select
“Count” and set “Peaks” to “3.” Set Precursor mass analyzer
to “Centroided” and set “Mass Accuracy” to “10.” Under the
bottom box “Retention time filtering,” set “Use only scans
within 5 min of MS/MS IDs” (see Note 4).
6. “Import FASTA” will appear. Set “Enzyme” to “trypsin” and
“Max missed cleavages” to “1.” Click “Browse. . .” to find the
database used for the MS-Fragger database search. Skyline will
now match the peptide identifications back to the proteins in
the database (see Note 5).
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Jesse G. Meyer
appropriate location (e.g., C:\FragPipe\tutorial_output\), and
then click “RUN” near the top left. The entire analysis workflow will now commence. This will take about an hour
depending on the speed of your computer. After successful
processing, you should find several new files in the folder you
specified including “combined.pep.xml,” which we will use in
the next section.
3.2 Quantification
of Peptides
and Detection
of Protein Changes
1. The combined peptide identifications from Subheading 3.1 are
imported into the Skyline software for peptide-level quantification. Open Skyline and select “Import DDA Peptide Search”
from the options on the start page. You will be prompted to
save the document.
2. In the “Import Peptide Search” box that pops up, change the
cutoff score to “0.99,” click “Add Files. . .” on the right side,
and select the output from FragPipe in the last section “combined.pep.xml.” Click “Next” to start building the spectral
library. This step will take about 10 min with the tutorial
data, and the duration is proportional to the number of confident peptide-spectrum matches produced by FragPipe/MSFragger analysis (see Note 3).
3. Once the library is finished building, the box will change to
“Extract Chromatograms,” and Skyline should find your
source mzML files and populate the “Results files found” list.
If your files are not in the list, browse the file system and add
them. Click “Next.” A box should pop up asking if you want to
remove the common file prefix. This makes viewing the replicates easier. Click “OK” to remove the prefix.
4. The box will change to “Add Modifications” and will show a
list of modifications found in the library that you can choose
to include in the quantification. For the purpose of our
analysis, check the box next to “Acetyl (N-term) ¼ [42]”
and “Oxidation (M) ¼ M[16],” and then click “Next >” at
the bottom.
5. “Configure Full-Scan Settings” will appear. Set precursor
charges to “2, 3, 4, 5.” Under “Isotope peaks included,” select
“Count” and set “Peaks” to “3.” Set Precursor mass analyzer
to “Centroided” and set “Mass Accuracy” to “10.” Under the
bottom box “Retention time filtering,” set “Use only scans
within 5 min of MS/MS IDs” (see Note 4).
6. “Import FASTA” will appear. Set “Enzyme” to “trypsin” and
“Max missed cleavages” to “1.” Click “Browse. . .” to find the
database used for the MS-Fragger database search. Skyline will
now match the peptide identifications back to the proteins in
the database (see Note 5).
302
Jesse G. Meyer