by the delimiter “|” into three new columns. To do this without
overwriting the adjacent columns, you will need to create two
empty columns to the right of the “Protein” column.
3. Open Cytoscape, and install the “ClueGo+CluePedia” plug-ins
using the “App Manager” under the “Apps” menu. This will
require registration of the app and may take a day or two to get
your license.
4. Open the ClueGO app in Cytoscape by selecting it from the
“App” menu.
5. Download the mouse (Mus musculus) ontologies. Under the
“ClueGO+CluePedia” tab that pops up under the control
panel at the left, click the button with a paw icon under
“Load Marker Lists” near the top. Select Mus musculus from
the list and click “download.” After the download, select “Mus
musculus” from the drop-down box next to the button with
the paw icon (see Note 6).
6. Copy the 6-character UniProt accessions with negative log2FC
values into the box near the top left under “Load Marker List
(s),” and then click the plus sign below the box to “Add an
additional input cluster.” Copy the 6-character UniProt accessions with positive log2FC values into the second box.
7. Under the next section “Visual Style,” select the middle option
“Clusters” to color the functions based on whether they are in
the up- or downregulated group.
8. Set the ontology options. Under “ClueGO Settings,” in the list
of “Ontologies/Pathways,” select “GO Biological Process” for
this initial analysis. About three-fourths of the way down the
options, click the box next to “Show only Pathways with
pV. . .,” and set the threshold to 0.00001. Keep all other settings as the defaults for this initial analysis, and click “Start” at
the bottom. This will produce a number of enriched GO
Biological Functions similar to Fig. 3 (see Note 7).
4 Notes
1. Ensure that experiment and replicate columns are populated. If
you skip this step, the program will not run iProphet to combine the search results from each of the files.
2. It is very important to check the boxes “Multi-experiment
report” and “Generate peptide level summary” under the
“Report” tab in FragPipe, because adding these options
ensures that iProphet is run to combine the individual peptide
searches in a statistically rigorous way.
Qualitative and Quantitative Shotgun Proteomics Data Analysis. . .
305
overwriting the adjacent columns, you will need to create two
empty columns to the right of the “Protein” column.
3. Open Cytoscape, and install the “ClueGo+CluePedia” plug-ins
using the “App Manager” under the “Apps” menu. This will
require registration of the app and may take a day or two to get
your license.
4. Open the ClueGO app in Cytoscape by selecting it from the
“App” menu.
5. Download the mouse (Mus musculus) ontologies. Under the
“ClueGO+CluePedia” tab that pops up under the control
panel at the left, click the button with a paw icon under
“Load Marker Lists” near the top. Select Mus musculus from
the list and click “download.” After the download, select “Mus
musculus” from the drop-down box next to the button with
the paw icon (see Note 6).
6. Copy the 6-character UniProt accessions with negative log2FC
values into the box near the top left under “Load Marker List
(s),” and then click the plus sign below the box to “Add an
additional input cluster.” Copy the 6-character UniProt accessions with positive log2FC values into the second box.
7. Under the next section “Visual Style,” select the middle option
“Clusters” to color the functions based on whether they are in
the up- or downregulated group.
8. Set the ontology options. Under “ClueGO Settings,” in the list
of “Ontologies/Pathways,” select “GO Biological Process” for
this initial analysis. About three-fourths of the way down the
options, click the box next to “Show only Pathways with
pV. . .,” and set the threshold to 0.00001. Keep all other settings as the defaults for this initial analysis, and click “Start” at
the bottom. This will produce a number of enriched GO
Biological Functions similar to Fig. 3 (see Note 7).
4 Notes
1. Ensure that experiment and replicate columns are populated. If
you skip this step, the program will not run iProphet to combine the search results from each of the files.
2. It is very important to check the boxes “Multi-experiment
report” and “Generate peptide level summary” under the
“Report” tab in FragPipe, because adding these options
ensures that iProphet is run to combine the individual peptide
searches in a statistically rigorous way.
Qualitative and Quantitative Shotgun Proteomics Data Analysis. . .
305