Do we need to add the iRT standards to each fractions when building the library?

Do we need to add the iRT standards to each fractions when building the library? zzhang9  2019-01-21

Hi there,

I am new to DIA and I have a question about the iRT values. When we building the library, we usually fractionate the sample and run each fraction individually in DDA mode. However, when we do database search using PD or MaxQuant, we only get one file. How could they use the iRT peptides' retention times in each fraction to calculate the iRT values? Thank you!


Brendan MacLean responded:  2019-01-21

Yes. Put your iRT standards into every sample. Regardless of whether the search result format batches many files together or keeps them separate, Skyline will figure this out and correctly calculate iRT values for your library.

zzhang9 responded:  2019-01-22

Hi Brendan,

I am curious how to assign the iRT standards' retention time to each fraction? how could the software know which iRT standards' retention time should be used to calculate the iRT values of the peptides in a specific fraction?

I have a set of standards which I want to use as iRT calculation. However, my standards can't be fused into a single sequence and identified during database searching. Do you have any suggestions how to solve this problem? Thank you so much!



Brendan MacLean responded:  2019-02-14

Hi Zhenbin,
What are your standards? Why can't you create a single contrived sequence that will produce them all from in silico digestion? Are they non-tryptic and you otherwise want to use tryptic digestion? Are you saying that your PD and MaxQuant searches are not currently finding all of your iRT standards? You will really need matched MS/MS spectra to your iRT standards in every run, if you want the most automatic support in Skyline to work for you.

Alternatively, if you can build up a fairly comprehensive iRT library before you try to import your searched fractions, if Skyline fails to find enough information about the standards in your library but is able to find enough run-to-run overlap (possibly challenging with fractions) and library-to-run overlap, it will use a regression between matched peptides and (non-standard) library peptides to derive iRT values, which just need to be derived relative to other peptides in our library.

If you can provide more information or example files, I may be able to help more.


anatoly.urisman responded:  2019-04-20

Hi Brendan,
I have a question related to this thread and your last paragraph above. I want to use endogenous peptides as iRT and a heavily fractionated library, in which individual fractions do not contain the full set of iRT peptides. It looks like when I attempt to add such a library to the iRT database, Skyline tries to find the iRT peptides in each of the fractions to align them individually rather than aligning the library based on run-to-run overlap first and then to iRT. Am I correct on this? If yes, is there a suggested work-around for this particular situation?