7. “Import FASTA” dialog box will change to show the number
of the proteins, peptides, precursors, and transitions detected
during the data import. If you followed the tutorial data up to
this point, it should say something like “This operation has
created the following targets: 7788 proteins, 63,342 peptides,
75,126 precursors, 225,378 transitions.” At this step, you have
the option to filter the proteins by the number of peptides per
protein and whether the peptides are unique for a single protein. For the purpose of the tutorial including downstream
peptide correlation analysis, we will only keep proteins with at
least 2 peptides, but for your own data, it may be acceptable to
keep proteins with 1 peptide. Check the bottom box “Remove
duplicate peptides,” which will remove peptides that match
multiple proteins. Peptides that are not unique to one protein
can confuse quantification because their quantity results from
multiple sources. For the tutorial data, these filters should
reduce the data to about 2918 proteins and 27,685 peptides.
Once you click “Finish,” Skyline will start extracting the peptide signals for all peptides in the document and all 15 files.
This step will take time depending on your computer’s processor speed. After this step completes, save the document to
ensure you keep your progress.
8. Prepare the document for MSstats comparison by adding the
“Condition” and “BioReplicate” annotations. From the “Settings” menu, select “Document Settings.” Under the “annotations” tab, click “Add. . .” on the right side. Under “Name:,”
type “Condition,” set the “Type” drop-down to “Text,” and at
the bottom under “Applies to,” check the box next to “Replicates.” Click “OK.” Repeat the same steps to add an annotation named “BioReplicate” with the same settings. Ensure that
the boxes next to your new annotation categories are checked
in the “Annotations” tab of the “Document Settings” window
and then click OK.
9. Assign values to the annotations you added in the previous
step. Go to “View” > “Document Grid.” Within the Document Grid window, at the top left, select “Reports” > “Replicates.” Add the annotations for “Condition” and
“BioReplicate” that match the file names (if using the tutorial
data). For example, for the file named “50mM_EtOH_3,” type
“EtOH” in the condition column, and type the appropriate
replicate number “3” under the “BioReplicate” column.
10. Install the MSstats tool by going to “Tools” > “Tool Store. . .”
and selecting MSstats. Follow the prompts to install MSstats.
11. Run MSstats analysis to determine protein quantity changes.
From the “Tools” menu, select “MSstats” > “Group Comparison.” Skyline will take a moment to write a report table for
Qualitative and Quantitative Shotgun Proteomics Data Analysis. . .
303
of the proteins, peptides, precursors, and transitions detected
during the data import. If you followed the tutorial data up to
this point, it should say something like “This operation has
created the following targets: 7788 proteins, 63,342 peptides,
75,126 precursors, 225,378 transitions.” At this step, you have
the option to filter the proteins by the number of peptides per
protein and whether the peptides are unique for a single protein. For the purpose of the tutorial including downstream
peptide correlation analysis, we will only keep proteins with at
least 2 peptides, but for your own data, it may be acceptable to
keep proteins with 1 peptide. Check the bottom box “Remove
duplicate peptides,” which will remove peptides that match
multiple proteins. Peptides that are not unique to one protein
can confuse quantification because their quantity results from
multiple sources. For the tutorial data, these filters should
reduce the data to about 2918 proteins and 27,685 peptides.
Once you click “Finish,” Skyline will start extracting the peptide signals for all peptides in the document and all 15 files.
This step will take time depending on your computer’s processor speed. After this step completes, save the document to
ensure you keep your progress.
8. Prepare the document for MSstats comparison by adding the
“Condition” and “BioReplicate” annotations. From the “Settings” menu, select “Document Settings.” Under the “annotations” tab, click “Add. . .” on the right side. Under “Name:,”
type “Condition,” set the “Type” drop-down to “Text,” and at
the bottom under “Applies to,” check the box next to “Replicates.” Click “OK.” Repeat the same steps to add an annotation named “BioReplicate” with the same settings. Ensure that
the boxes next to your new annotation categories are checked
in the “Annotations” tab of the “Document Settings” window
and then click OK.
9. Assign values to the annotations you added in the previous
step. Go to “View” > “Document Grid.” Within the Document Grid window, at the top left, select “Reports” > “Replicates.” Add the annotations for “Condition” and
“BioReplicate” that match the file names (if using the tutorial
data). For example, for the file named “50mM_EtOH_3,” type
“EtOH” in the condition column, and type the appropriate
replicate number “3” under the “BioReplicate” column.
10. Install the MSstats tool by going to “Tools” > “Tool Store. . .”
and selecting MSstats. Follow the prompts to install MSstats.
11. Run MSstats analysis to determine protein quantity changes.
From the “Tools” menu, select “MSstats” > “Group Comparison.” Skyline will take a moment to write a report table for
Qualitative and Quantitative Shotgun Proteomics Data Analysis. . .
303