2.3 Gene Ontology
Term Enrichment
Analysis and Network
Visualization
1. Cytoscape network analysis software (latest version, used 3.5.1
here, https://cytoscape.org/) [15].
2. Cytoscape plug-in ClueGO (latest version, download within
Cytoscape from App Manager, http://apps.cytoscape.org/
apps/cluego) [16].
3 Methods
3.1 Peptide
Identification
by Database Search
1. Download the tutorial MS files and install the software listed in
Subheading 2.
2. Convert. RAW files to mzML format using MSconvert. Right
click on one. RAW file, and then select “Open with MSconvertGUI.” At the top left, click “Browse,” and add all the files.
Under “Options” in the bottom left, set the output format to
“mzML,” and the “Binary encoding precision” to 64-bit.
Under “Filters” on the right, from the top drop-down menu,
select “Peak Picking” and click “Add” below. There should be
two filters in the box at the bottom right as pictured in Fig. S1.
NOTE: Ensure you have at least an additional 25 GB of space
on the drive that holds your mzML files.
3. Open the FragPipe.exe and start with the first tab named
“Config.” You will notice pop-up prompts asking you to
enter the location of the MSFragger.jar file and the Philosopher.exe file. Click browse for each of these and navigate to the
location of each of these.
4. Under the second tab “Select LC/MS Files,” click “Add files”
at the top left, and navigate to the mzML files you created in
the first step. Add group names to the “Experiment” column
and replicate numbers to the “Replicate” column matching the
file names (see Note 1).
5. On the third “Database” tab, click “Download” on the top
right to download the mouse database (if using the tutorial
data or your data is from mouse). In the pop-up box, click the
box for “add isoforms,” and then select “Mus musculus” below,
and then click “Yes” at the bottom to start downloading the
FASTA database.
6. On the fourth tab “MSFragger,” near “Load defaults” at the
top, click “Closed Search” to load the default parameters.
Under “Peak Matching” near the top, change the precursor
mass tolerance to À20 and 20.
7. Skip the fifth tab, and on the sixth “Report” tab, under the top
“Report” group at the top, check the box next to “Generate
peptide level summary.” In the middle group “Quantitation,”
uncheck the box “Run Quantitation” (see Note 2).
Qualitative and Quantitative Shotgun Proteomics Data Analysis. . .
301
Term Enrichment
Analysis and Network
Visualization
1. Cytoscape network analysis software (latest version, used 3.5.1
here, https://cytoscape.org/) [15].
2. Cytoscape plug-in ClueGO (latest version, download within
Cytoscape from App Manager, http://apps.cytoscape.org/
apps/cluego) [16].
3 Methods
3.1 Peptide
Identification
by Database Search
1. Download the tutorial MS files and install the software listed in
Subheading 2.
2. Convert. RAW files to mzML format using MSconvert. Right
click on one. RAW file, and then select “Open with MSconvertGUI.” At the top left, click “Browse,” and add all the files.
Under “Options” in the bottom left, set the output format to
“mzML,” and the “Binary encoding precision” to 64-bit.
Under “Filters” on the right, from the top drop-down menu,
select “Peak Picking” and click “Add” below. There should be
two filters in the box at the bottom right as pictured in Fig. S1.
NOTE: Ensure you have at least an additional 25 GB of space
on the drive that holds your mzML files.
3. Open the FragPipe.exe and start with the first tab named
“Config.” You will notice pop-up prompts asking you to
enter the location of the MSFragger.jar file and the Philosopher.exe file. Click browse for each of these and navigate to the
location of each of these.
4. Under the second tab “Select LC/MS Files,” click “Add files”
at the top left, and navigate to the mzML files you created in
the first step. Add group names to the “Experiment” column
and replicate numbers to the “Replicate” column matching the
file names (see Note 1).
5. On the third “Database” tab, click “Download” on the top
right to download the mouse database (if using the tutorial
data or your data is from mouse). In the pop-up box, click the
box for “add isoforms,” and then select “Mus musculus” below,
and then click “Yes” at the bottom to start downloading the
FASTA database.
6. On the fourth tab “MSFragger,” near “Load defaults” at the
top, click “Closed Search” to load the default parameters.
Under “Peak Matching” near the top, change the precursor
mass tolerance to À20 and 20.
7. Skip the fifth tab, and on the sixth “Report” tab, under the top
“Report” group at the top, check the box next to “Generate
peptide level summary.” In the middle group “Quantitation,”
uncheck the box “Run Quantitation” (see Note 2).
Qualitative and Quantitative Shotgun Proteomics Data Analysis. . .
301