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Display of protein abundances in PRM experiment

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Display of protein abundances in PRM experiment bpfister  2021-02-05 02:55
 

Dear Skyline team,

I was very excited about the recent implementation to show the actual protein abundances of each replicate within the Group Comparison grid, but am having problems with displaying them correctly. As my experiment included spiked-in heavy labelled peptides, I used "ratio to heavy" as the normalization method for the group comparison (see screenshot). I understood that I should also apply this normalization method in Peptide Settings --> Quantification (screenshot), but when I do that, it will only display me N/A for all replicates and proteins in the Group Comparison grid (why?).
When using "Equalize medians" in Peptide Settings --> Quantification, the protein abundances are displayed but they don't give me values that would lead to the fold changes calculated by Skyline (also not if I change the normalization to "Equalize medians" for the group comparison). Which settings do I need to use to get exactly the data that underlies the fold change calculated by Skyline?

I am using Skyline version 20.2.0.343.

Thanks a lot for your help!
Barbara
 
 
Nick Shulman responded:  2021-02-05 07:11
The protein abundance is supposed to be #N/A if any of the replicates have missing values.

However, that logic is not supposed to be applied if your normalization method is ratio to heavy, then that is not supposed to be #N/A.

Skyline 20.2 has a bug in it where that logic got reversed: Ratio to heavy is the _only_ normalization method which does _not_ allow for missing values. (and this is the incorrect behavior)

This bug was first reported in October:
https://skyline.ms/announcements/home/support/thread.view?rowId=48841

Thank you for reminding me. I will make sure to fix this in Skyline-Daily soon.
-- Nick
 
bpfister responded:  2021-02-05 07:32
Dear Nick,

thanks a lot for your fast and helpful reply. I will wait for the updated Skyline-Daily version then.

Best,
Barbara
 
Nick Shulman responded:  2021-02-05 09:02
By the way, in the current Skyline-Daily, you can actually see the protein abundance which was used to calculate the fold changes. This abundance value does not have this bug, and will not be #N/A for you.
This abundance value is available if you choose the report "Clustered" in the Reports dropdown on Group Comparison grid. This abundance value will be calculated using the normalization method that was chosen for the group comparison.

This abundance value was added as part of the new "hierarchical clustering" feature. You can read more about this feature here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=HierarchicalClustering
(We would appreciate any feedback that you might have about this new feature)
-- Nick