In the Internal Standard Concentration column, add the known amount of fmol for each heavy labeled peptide (see the annex at the end of this tutorial). Now, again from the “Document Grid” window Go to “View” “Peptide Ratio Results”. Now, in the quantification column you have the amount of each endogenous peptide calculated with the single point calibration method. Now the “Document grid” should look like this: Statistical comparison between conditions Annotating samples with group information To perform statistical analysis of the results we first need to annotate which samples are replicates. Skyline allows you to associate additional information with the runs in the document by defining custom annotations. To view the Annotation Settings form, perform the following steps: On the “Settings” menu, click “Document Settings”. In the “Annotations” Tab click “Add”. Name the new “Annotation” as “Condition” Type: Value List Values: “G1”, “G2M”, “S”, each on a new line (without “ ”) Applies To: “Replicates” Click “OK”. Generate another “Annotation” named “BioReplicate” following the same steps as above. Name the new “Annotation” as “BioReplicate” Type: Value List Values: “1”, “2”, “3”…”9” each on a new line (without “ ”) Applies To: “Replicates” Click the “OK” button. Select both “Annotations” in the “Annotations” tab. Editing the annotation values in Skyline is done using the “Document Grid”. To bring up the Document Grid go to the “View” menu and click “Document Grid Go to “View” → ”Replicates” Annotate “Condition”, “BioReplicate” as shown in the table below. Close the Document Grid Save the Skyline file as PRM_Annotated.sky in Skyline Webinar folder.
Group Comparison in Skyline Skyline can perform pairwise group comparisons of peptide and protein peak areas. The comparisons are performed by i) considering all the available transition peak areas for a peptide or protein, ii) optionally dividing by a normalization standard, iii) taking the log, iv) averaging any technical replicates and v) performing a t-test on the resulting values. Skyline automatically discards replicates with missing values. To perform the group comparisons follow the next steps: On the “Settings menu”, click “Document Settings”. Go to the “Group Comparisons” tab and Click the “Add” button of the Document Settings form. In the Name field of the Edit Group Comparison form, enter “G2M-vs-G1”. For the Control group annotations choose “Condition”. For the Control group value choose “G1”. For the Value to compare against choose “G2M”. For the Identity annotation choose “BioReplicate”. For the Normalization method choose “Ratio to Heavy”. In the Confidence level field, enter “95” %. For Scope choose Protein. Click the “Advanced” tab and select “Tukey’s Median Polish” in the drop down menu of the “Summary Method” The “Edit Group Comparison” form should look like this: Click the “OK” button. Perform the same steps to configure a Group Comparison called “S-vs-G1”. We will choose in this comparison for the Value to compare against “S”. To inspect the group comparison you just defined do the following: On the View menu, choose Group Comparisons and click “G2M-vs-G1” or Skyline will show a grid view that looks like this: Be aware that numbers can be different in your document depending on which peptides/transitions you have selected for the refined file. In the “G2M-vs-G1” comparison: Click the “Bar Graph” button in the menu above the toolbar in the grid view. Skyline adds a graph tab to the view that looks like this: On the grid, click the “Fold Change Result” header, and click Sort Ascending in the menu that appears. This will sort the grid, and if you switch back to the graph, you will see that it is also sorted.