Are all peptides incl. iRTs used for normalization?

Are all peptides incl. iRTs used for normalization? roberthardt  2022-05-31

Dear Skyline-team,

I am just curious to know if Skyline actually uses all peptides/precursors present in the document when I select "normalize medians" in the quantification settings? Or does it only use the target peptides and omits the iRTs? I am asking because I currently rely on the CiRt peptides, of which not all are present in all my samples at the same amounts. Thus taking also this peptides into account for normalization could potentially skew my results.



Nick Shulman responded:  2022-05-31
iRT standards are not included.
Other sorts of standards (Global Standard, QC, and Surrogate Standards) are included in the median calculation.
Note that median normalization is only valid to be used if the number of peptides in your Skyline document which you expect to not be changing is much larger than the number of peptides which might be changing.
For instance, if you Skyline document had only one peptide in it, and you were to use median normalization, you would expect to find that the fold change between any two replicates would be 1.

By the way, here is the code where Skyline calculates the median of the data in all of the replicates:
On line 66, you can see that Skyline skips peptides whose Standard Type is "iRT".
-- Nick