Fold change

Fold change ilaria palmisano  2024-01-04 07:22

Dear all,
I am a beginner and I have a question about how skyline calculates the Fold Change between groups. I understood that, asking a Clustered report, one can get the protein abundance values used to calculate the FC. But, using those values, I am not able to reproduce the FC calculated by Skyline.
Could you please help me? Thanks

Nick Shulman responded:  2024-01-04 08:06
Skyline performs a linear regression in order to turn those protein abundance values into a fold change.
There is an explanation of how that linear regression works in my October 17, 2019 response here:

If you still have questions about how Skyline is calculating fold changes in your document, you could send us your Skyline document.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
If that .zip file is less than 50MB you can attach it to this support request. You can upload larger files here:
-- Nick
ilaria palmisano responded:  2024-01-04 09:23
Thank you.
How can I retrieve the values used to calculate the Fold change, please? Is there any way to retrieve it form the report? I think that can help me understand better.
Thanks a lot
Nick Shulman responded:  2024-01-04 09:46
The "Abundance" values that you see when you choose "Clustered" in the "Reports" dropdown at the top of the Group Comparison grid are the numbers that get used in the linear regression to calculate the fold change.

-- Nick
ilaria palmisano responded:  2024-01-04 10:28
Thank you. Is the FC calculated by performing the ratio between the "abundance" values between the 2 groups? I am not able to reproduce the FC.

I am doing the following:
-calculate the average of the abundance values for each group (6 replicates)
-calculate the ratio of the averaged abundance values

I am not sure if I am doing something incorrect.
Nick Shulman responded:  2024-01-04 10:46
No, the fold change is calculated by plotting the logarithm of the abundance values as the y-coordinates on a graph, with the x-coordinates being either 0 or 1 depending on whether the Replicate is in the Control group or the other group.

After these values have been plotted on the graph, a linear regression is performed, and slope of that linear regression is the logarithm of the fold change.

The method was described in this post:
There is also an Excel spreadsheet attached to that post which does the calculation in Excel using the "LINEST" function.

If you had only one replicate in each group, of if the "Identity Annotation" was specified, and there was only one unique value for the Identity Annotation in the two groups, then the linear regression method would result in the same number as taking the averages and dividing. But, if you have more than one replicate, then the linear regression yields a different number, probably because the linear regression is trying to minimize the square of the distance of the points to the line.
-- Nick
ilaria palmisano responded:  2024-01-04 11:08
Thanks a lot! All clear now!