Isolation List for Heavy Labeled Peptide with No Data philip remes  2023-04-24 13:36
 

Hi,

Problem: I have data for endogenous peptides and want to export an isolation list that includes both heavy and light peptides. However, Skyline does not let me export the isolation list, saying that I need to either select a retention time predictor or else import results for all the peptides. I think that actually Skyline has all the information it needs, since the light and heavy peptides have the same retention times. Do you agree, and if so, could this be added as a feature?

More Details:
I have a Skyline document where a Proteome Discoverer result was imported. Offline I compared the results to a list of heavy labeled peptides, and then filtered the results with Refine/Accept Peptides. Then I added the R/K heavy modification option from Settings/Peptide Settings/Modifications. I select Export Isolation List/Thermo Fusion Tune +3 with Scheduled Times.

Thanks
Philip
 
 
Nick Shulman responded:  2023-04-24 14:08
Can you send us your Skyline document?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including iRT databases, libraries and extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request.
You can upload larger files here:
https://skyline.ms/files.url

The way that this is supposed to work is that the iRT database is only supposed to contain light peptide sequences.
One way that it would be possible to confuse Skyline would be to go to "Settings > Peptide Settings > Modifications" and add an isotope modification to the upper list box which is supposed to only contain structural modifications.
We also have had bugs at other times where Skyline was either storing a heavy modification in the iRT database or was trying to look for a heavy modification.
After we see your Skyline document we will probably be able to figure out what is going wrong.
-- Nick
 
philip remes responded:  2023-04-24 15:00
Hi Nick,

Thanks for getting back to me. As I was preparing the document for you, I realized that I couldn't actually export a regular isolation list either, with all light peptides. The problem was that there was one peptide somewhere in the document that already had no data, that I couldn't see because there's so many proteins. When I used Refine/Remove Missing Results, then I could export the isolation list for light peptides, and also for the light/heavy peptides after added the modifications. Sorry for the mixup.

Thanks
Philip
 
Nick Shulman responded:  2023-04-24 15:47
It sounds like maybe we should change the error message that you saw so that it tells you which peptide does not have retention time information.
If you feel like it, it might still be helpful if you could send us the document that was causing you trouble.
-- Nick
 
philip remes responded:  2023-04-25 07:33
Hi Nick,

I put the document in the share location, with the name 230424_export_heavy_for_skyline.sky.zip. Just delete it from the share location after downloading if you please. It's in the state where you'll get the error message if you try to export.

Thanks
Philip