PRM assay missing peptides when I create a big library from all my raw files

PRM assay missing peptides when I create a big library from all my raw files s0dharor  2023-03-28


So I'm working on developing a big assay for targeting ~300 proteins. During the peptide selection process , the libraries were created with PRM raw files using Proteome discoverer. Now, when I'm merging this big assay with all the individual libraries. Some peptides are missing the dotp values eventhough they had it earlier. So basically I wanna know how can my peptides be assigned a library spectrum,

Nick Shulman responded:  2023-03-28
One thing that might be going on is that your Skyline document does not have enough MS2 transitions for the particular precursors that are missing library dot product values.
Skyline requires at least 3 transitions in order to calculate dot products.

Otherwise, I am not sure what might be going on. If you would like, you can 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 spectral 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:

-- Nick
s0dharor responded:  2023-03-29
Hi Nick,

Thanks for your reply!

I think the files are too large to be uploaded (30.5 GB). Is there any other alternative?
s0dharor responded:  2023-03-29
And additionally when I'm trying to share a file it gives an error saying : ystem.IO.IOException: Settings.MeasuredResults No final cache
Settings.PeptideSettings.Libraries: null library
   at pwiz.Skyline.SkylineWindow.ShareDocument(String skyZipFileName) in C:\proj\pwiz_x64\pwiz_tools\Skyline\SkylineFiles.cs:line 1192
Nick Shulman responded:  2023-03-29
I believe the error "No final cache" that you are seeing might mean that Skyline has not finished extracting chromatograms.
Does the status bar at the bottom of the Skyline window give you any indication that Skyline is still doing work?

It might be that there is some reason that Skyline has not finished doing that, such as that Skyline was unable to load all the libraries.
In cases where Skyline was unable to completely load the document, you are unfortunately unable to use the "File > Share" menu item.

In cases like these, you might want to just send us as many files as you can.
If you use the menu item:
File > Open Containing Folder
the folder containing the Skyline document will open in Windows Explorer, with the Skyline document file selected.
If you send us that Skyline document file (it has the filename extension ".sky") I could then tell you the names of the rest of the files that I would need to reproduce this problem.

Feel free to send us 30GB of files if you would like. We have plenty of disk space on the server that you would be uploading to.
-- Nick
s0dharor responded:  2023-03-30
Hi Nick,

So while transferring files from different computers to upload. I get another error which is ---------------------------
Failure extracting Skyline document from zip file Z:\AG Winter\PEOPLE\Dhriti Arora\DA_YH_PRM assay\
Bad state (invalid literal/length code). I would also like to know why this is.


Nick Shulman responded:  2023-03-30
That error looks like an error that you would get if the data in the .zip file was different than what it was supposed to be (i.e. the .zip file is corrupted).
You might be able to fix the problem by creating the .zip file again.
-- Nick