No data import when PROSIT generated library is activated pavel shliaha  2022-04-08 05:44
 

I am trying to load in some data against a library generated in PROSIT. However I get an empty chromatogram with RT from 0-1.2min, as shown in slide 1 (my runs are 0-80 min). If I then Go to peptide settings > library and untick the library the runs load normally, as shown in slide 3, but of course dotp cannot be calculated. Any suggestions why? Note in the library the predicted RT are all over 80 min (slide 4). Could this be a problem?
 
 
Nick Shulman responded:  2022-04-08 06:34
One thing that might be going on is that your settings are telling Skyline to only extract chromatograms within a certain distance of the predicted retention time.
You should go to:
Settings > Transition Settings > Full Scan
and at the bottom there is the "Retention time filtering" setting. You might need to change that to "Include all matching scans"

Other than that, I am not sure what is going wrong. You should send us your Skyline document and one of your raw files.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including whatever chromatograms have been extracted.

If that .zip file and your raw file are less than 50MB you can attach them to this support request. You can upload larger files here:
https://skyline.ms/files.url
-- Nick
 
pavel shliaha responded:  2022-04-08 07:03
yep. Thanks it worked! If you have a second, could you please tell me where the dotp filtering functionality is. E.g. is it possible to ask skyline to pick the peak with highest dotp or to introduce a product, that demands at least 0.75 dotp for a peak to be called?
 
Nick Shulman responded:  2022-04-08 07:30
The library dot product is part of the scoring function that Skyline uses to decide which peak to pick.
There is no way to tell Skyline to only pick peaks with a dot product above a certain value. The dot product score is multiplied by its assigned weight and added to the other parts of Skyline's scoring function.

If you want to see the list of feature scores that Skyline looks at in its default peak scoring model you can go to:
Refine > Reintegrate
and then choose "<Add...>" in the Peak scoring model dropdown.
Then, in the "Choose model" dropdown on the Edit Peak Scoring Model form, choose "Default". You will then see the list of the seven feature scores that Skyline uses in its default peak scoring model.

If you have a dataset with decoy peptides in it you can tell Skyline to train a new peak scoring model. You can learn how to do that in the Advanced Peak Picking Models tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_peak_picking

-- Nick