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How to analyze prm-PASEF data with Skyline using spectral library exported from Spectronaut and bypass iRT

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How to analyze prm-PASEF data with Skyline using spectral library exported from Spectronaut and bypass iRT boxi  2025-02-27 17:25
 

Hello Skyline Team,
We want to use Skyline to analyze prm-PASEF data acquired from timsTOF HT. We first used Spectronaut to generate the spectral library from DDA-PASEF data (we do not have software to directly analyze DDA-PASEF data) and then exported the spectral library tsv file. As previous post suggested, we imported the spectral library tsv file through "Assay Library". However, it requires us to have iRT information to generate the library. Is there a way we can bypass iRT option since our goal is not for absolute quantification and we do not spike in iRT standards? We also tried to choose a protein from our target list as a standard, but in order for it to work, all the peptides from the chosen protein has to be presented in our acquired prm data, but we only want to monitor 2-3 major peptides, so this is not really work for us. Do you know how can we solve this issue? Is there another way to analysis the prm-PASEF data without using iRT information? Thanks!
 
 
Nick Shulman responded:  2025-02-27 22:26
I am not aware of any reason that you would need to have iRT when creating a library.
There is a dropdown on the first page of the "Import Peptide Search" or "Build Library" wizards where you can choose "iRT standard peptides", but you can leave that set to "None".
Can you send us a screenshot of what you are seeing?

It might also help if you sent us the files that you are working with.

Files which are less than 50MB can be attached to these support requests.
If your files are larger than that you can zip them up and upload them here:
https://skyline.ms/files.url
-- Nick
 
boxi responded:  2025-02-28 09:25
Hello Nick,
Thanks for your reply. We use the output from Spectronaut. According to your team, the file export from Spectronaut can not use "Import Peptide Search" or "Build Library" but should import through "Assay library" which I did not find a way to bypass iRT.
Please see attachments for our issue.
Screenshot 00: The import method we use to import tsv file exported from Spectronaut to built library.
Screenshot 01: Error message if we did not define iRT from the import file.
Screenshot 02: We tried to defined iRT using one of our target protein and generated the library, however, when we imported the prm-PASEF data, we got this message said all standard peptides need to present in our data.
I also uploaded our prm-PASEF data and the tsv file exported from Spectronaut (zip file name: BOXI_prm-PASEF_support).
Thanks!
 
Nick Shulman responded:  2025-02-28 14:24
Thank you for uploading those files.
Your .tsv file did have iRT values in it.
When I tried to import it into Skyline as an Assay Library, Skyline complained that it did not have an ion mobility units column. I used Microsoft Excel to add a column "Ion Mobility Units" where every value was "1/K0 (Vs/cm^2)". (attached file "AssayLibrary.tsv")

Skyline did force me to choose a set of iRT standards when I imported "AssayLibrary.tsv".
If you do not want to use iRT with your document you can go to the "Prediction" tab at "Settings > Peptide Settings" and change the Retention Time Predictor to "None".
You might also have to go to the "Full Scan" tab at "Settings > Transition Settings" and make sure the retention time filtering option at the bottom is "Include all matching scans".

If you set the Retention Time Predictor to "None" then Skyline will not give you that error message about missing iRT standards.

I was able to extract chromatograms from your .d folder and I am attaching my Skyline document "test2.sky.zip".
-- Nick
 
boxi responded:  2025-02-28 14:45
Hello Nick,
Thanks a lot for your help! It works now.
I have another question. Is it possible to view the mobility information for each target peptide similar to the retention time graph?