iRT error when adding Protein Prospector generated blib

iRT error when adding Protein Prospector generated blib anatoly.urisman  2019-10-16

Dear Skyline team,
I am getting an error while adding an “on-column” spectral library (blib generated directly from Protein Prospector) to an iRT calculator (Pierce iRT set supported by Skyline). The error is “1 run was not converted due to insufficient correlation.”

I’ve uploaded a dummy Skyline document to the Upload folder. It contains two libraries, both of which are generated from the same DDA data, just two different searches (one focused on identifying the iRT peptides and the other on other peptides in the samples).

Could you take a look at the error and let us know if it is something that needs to be fixed on the Protein Prospector end?

Nick Shulman responded:  2019-10-16

I tried doing the following:
File > Import > Peptide Search
and point Skyline at the file "Pau_2019-07-10_iRT-Pierce.blib" that you have included in the .zip file you uploaded.

In the Import Peptide Search wizard when I chose "Pierce" as the iRT Standard Peptides that I had.

Skyline failed to do the linear regression because Skyline could not find any of the standards in that .blib file. If you click on the "failed" link on the "Add iRT Peptides" dialog you will see that all of the standards are "Missing".

I can tell you that the reason that Skylne cannot find the standards in the .blib file is that Skyline is looking for the unmodified forms of the peptides (e.g. "SSAAPPPPPR") and what is actually in the .blib file is modified (i.e. "SSAAPPPPPR[+10.0]").

This seems like a Skyline bug but I will have to ask around tomorrow to be sure.

There are other places in Skyline where Skyline is smart enough to find heavy modified forms of peptides in spectral libraries if the light form is not present. I am not sure whether it was an oversight or by design that Skyline is not doing this during the iRT regression part of importing peptide search results.
-- Nick
anatoly.urisman responded:  2019-10-16
Hi Nick,
Thank you for looking into this. I am getting a similar error when I add the same library to the iRT calculator ( which is made from the Pierce preset in Skyline and appears to have the correct isotope labeled peptides).
Kaipo responded:  2019-10-21
Hi Anatoly,
Thank you for reporting this bug - I will make a fix for this shortly.
Kaipo responded:  2019-10-28
This bug fix has been made and will be included in the next release of Skyline-daily.

anatoly.urisman responded:  2019-10-28
Thank you very much, Kaipo.
I've been getting a similar error with a set of endogenous iRT peptides and suspect that this fix will resolve it. Once the new Skyline daily is out, I will test both the Pierce and my endogenous iRT sets and will let you know if the error persists.
- Anatoly