Peak normalization with iRT

Peak normalization with iRT c frampas  2024-01-09 07:52


I was wondering if it was possible to use iRT for peak normalization? If so, could you explain how to go about it?
Due to a large number of samples, I had to divide them into 2 batches. Unfortunately, the mass spec had an increase of sensitivity and therefore I can't compare samples. Your help would be appreciated.

Many thanks,

Nick Shulman responded:  2024-01-09 08:01
I am not sure that I understand your question.
Are you asking whether it is possible to use your iRT standards as both iRT standard and as "Global Standards"?
Yes, it is possible to do that, but you have to have two copies of these peptides in your document.

Normally, when you right-click on a peptide in the Targets tree, you can choose "Set Standard Type > Global Standards" in order to specify that these peptides should be used when calculating the "ratio to global standards".
However, when those peptides are iRT standards, Skyline will not let you specify that they are Global Standards.
In order work around this limitation, you should select the iRT peptides in the Targets tree and then do "Edit > Copy" and then do "Edit > Paste".
You will then have two copies of those peptides in the Targets tree. The first copy of those peptides will be displaying the clock icon indicating that they are iRT standards. The second copy of those peptides will not have that clock icon, so you will be able to right-click on the second copy of the peptides and choose "Set Standard Type > Global Standards".

I hope this answers your question. Let me know if your question was actually about something else.
-- Nick
c frampas responded:  2024-01-09 08:10
Hi Nick,

Thank you for your quick response! Yes, you have answered the question, I was indeed trying to use the iRT standards as "Global Standards" and will try your instructions. Thank you very much.