|iRT regression based failure to import DIA runs||kguehrs||2020-07-09|
Dear Skyline team,
I know that similar issues were already discussed but I am somewhat frustrated and not sure how ti further proceed.
I did 6 DIA runs for 2 preparation schemes of 3 samples similar to 3 biological replicates for 2 conditions. I added the Biognosys 1 peptides iRT reference to all sample and also used the Biognosys 11 calculator in Skyline in the peptide settings.
Four of the six runs were imported without problems. The remaining two run were not imported with the failure message shown in one of the screenshots in the attached zip archive. I added 6 additional figures that show the regression line for each of the samples together with the extracted peaks for one of the Biognosys 11 reference. We use for some special reason rather complex gradients that are optimized for the main samples that are often measured and that might be one possible explanation for the deviations of the retention times of some of the iRT peptides, but this behaviour is shared wth all samples.
I have tried to use different iRT calculated based on the Biognosys 11 with several samples peptides added but without success. I have also tried to use a Biognosys reference with only 9 or 10 peptides but this did not result in successful import. I also reduced the transitions to 3 product ions only without success of reimport.
I have asked a colleague to use the dataset with Biognosys software and he managed to import the sampled without any problem. Therefore, I assume that the problem is in Skyline and you might think one more time about the idea of r*r>=0.99 for the iRT regression being the most appropriate setting for import of DIA data that are more noisy and not that simple as PRM chromatograms.
Another suggestion relates to the import procedure. As far as I understood from previous discussions of the import issue the r*r>=0.99 for the iRT regression is calculated at the beginning of the import process but the error message is only shown after finalizing data import. In my trials I always had to wait some 10 minutes (rather old computer) until I had to realize the failure. If my assumption of the process timing is correct it should be possible to give an early message and possibility that the user can cancel the process to change settings and retry more early.
Perhaps, you can supply some more ideas how to progress as I am running out of ideas for further steps.
Thank you for your good work to offer Skyline for the community.