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Protein abundance vmohanty  2024-07-12 08:26
 

Hi All,

I have acquired my PRM data and using Skyline for computing my Fold change using MS stats.

I have 2 peptides corresponding to one protein and would like to know the Fold change at the protein level.
I am getting a foldchange at peptide level. I would like to know how can i get it right at protein level. Do i have to do any offline calculation for protein level?

I followed the Tutorial 20_1, however, my data is displayed in Fold change of each peptide with Adjusted P-Values.

I am ready to share my Skyline document too. Could you please provide me with some ideas how to do so. Than would be very helpful

Thank you.
Varsha
 
 
Nick Shulman responded:  2024-07-12 09:34
When you edit the settings for the Group Comparison, there is a box that says "Scope" and the two choices in there are "Protein" and "Peptide".
If you choose "Protein" you will get fold changes at the protein level.
 
vmohanty responded:  2024-07-12 09:43
Hi Nick,

I did try that but its not providing any information!

Varsha
 
Nick Shulman responded:  2024-07-12 10:01
Can you send me 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.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
If your data is confidential you can email it to me directly.

There are a number of things that might be going wrong.
I suspect that replicates are being excluded because some of their transitions have truncated peak areas, and, at the protein level, there are no replicates that have valid data for all of the transitions.

After I see your Skyline document I will probably be able to figure out what is going wrong.
-- Nick
 
vmohanty responded:  2024-07-12 10:21
Sure, Can I have your email ID please.
I want to share directly with you.

Thank you.
Varsha
 
Nick Shulman responded:  2024-07-12 10:54
Thank you for emailing me your Skyline document.
I think the problem is that all of your peptides have been marked as "Surrogate Standards".
When Skyline is calculating fold changes for proteins, Skyline always skips peptides that have been designated as some sort of standard.
You should right-click on the peptides in the Targets tree and choose "Set Standard Type > None".
-- Nick