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 click “Settings menu”, “Document Settings”. Go to the tab and Click the of the Document Settings “Group Comparisons” “Add” button 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 to “Ratio Heavy”. In the Confidence level field, enter “95” %. For Scope choose Protein. Click the tab and select in the drop down menu of “Advanced” “Tukey’s Median Polish” the “Summary Method” The Group should look like this: “Edit Comparison” form Click the button. “OK” Perform the same steps to configure a Group Comparison called We will “S-vs-G1”. 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 or “G2M-vs-G1” 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 comparison: “G2M-vs-G1” Click the button in the menu above the toolbar in the grid view. “Bar Graph” Skyline adds a graph tab to the view that looks like this: On the grid, click the Change and click Sort Ascending in the “Fold Result” header, menu that appears. This will sort the grid, and if you switch back to the graph, you will see that it is also sorted.
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.
Webinar%2017%20%28Chiva%29.docx