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how to export spectronaut generated spectral library in to skyline

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how to export spectronaut generated spectral library in to skyline gupta  2025-09-26 05:31
 
Hey people,
I know there are a few posts regarding some suggestions and ideas to export spectronaut generated spectral library in to skyline. Briefly, I generated DIA dataset using samples where heavy labeled peptides (Lys8 or Arg 10) from EGFR protein were spiked-in in Hela peptides (Pierce Hela digests) on Bruker's timsTOF machine (30SPD/44min). I used spectronaut 20 to generate spectral library for me (attached here in .csv format) and I would like to like to import this library into skyline. I am unable to find the best and easy way to do so as Skyline errors out with the message "score types not loaded" (attached). It would be great if someone has experience with this and help me to import that library for downstream analysis
Thank you,
Rajat
 
 
Nick Shulman responded:  2025-09-26 08:41
You should use the "File > Import > Assay Library" to import the Spectronaut csv file.

However, before you do that, you need to edit the .csv file and add a column "IonMobilityUnits" where the value in each row is "1/K0 (Vs/cm^2)".
I have done that with the attached file "AssayLibrary.csv". I also renamed a couple of columns to make it easier for Skyline to figure out which column is the "Precursor m/z" and "Product m/z" columns.

The other thing that you should do is tell Skyline about the modifications that you are using, which, in addition to C[+57] are Oxidation (M), water loss, ammonia loss, and the K and R heavy modifications. See attached screenshot "ModificationSettings.png".
I also went to the "Instrument" tab at "Settings > Transition Settings" and changed the Max m/z to 2000.

After I did all that I was able to import AssayLibrary.csv and I got the attached Skyline document "ImportedAssayLibrary.sky.zip".
Nearly all of the transitions were successfully imported but Skyline did give me 20 warnings. I think those warnings are cases where there was a neutral loss that Skyline does not think would be possible. For example, my settings said that a water loss could only be applied to D, E, S or T, whereas on line 987 of the CSV file it wanted to have a water loss on a y3 ion of ACGADSYEMEEDGVR.
You should probably ignore those small number of warnings.
(If you get significantly more than 20 warnings there's probably something wrong with your peptide modification settings)
-- Nick