Importing Spectranaut generated DIA-PASEF spectral library

Importing Spectranaut generated DIA-PASEF spectral library susmitaghosh191996  2023-03-28

Dear Team,

I have a problem in importing the DIA PASEF spectral library that is generated in Spectranaut version 17. While importing the .csv file in Skyline as assay library, it's giving some errors (attached below).I have attached the library as well for your reference.

Looking froward to your answer.

Brian Pratt responded:  2023-03-28

It looks like the attachments did not come through. Try again?

Thanks for using the Skyline support board,

Brian Pratt

Nick Shulman responded:  2023-03-29
Thank you for uploading your .csv file and the screenshot of the warning message that you got.
The warning message says things such as "Product m/z value 574.3348 in peptide ALGFLVR has no matching product ion" means that the CSV file said that that peptide should have a transition with that m/z but Skyline could not figure out what the identity of that transition is.
When I look at your CSV file, I see that the row with that peptide sequence and product m/z says "NH3" in the "Fragment Loss Type" column.

To fix that error, you should go to:
Settings > Peptide Settings > Modifications
and push the "Edit List" button next to the Structural Modifications list box then push "Add" and add the modifications with the following names:
Acetyl (N-term)
Ammonia Loss (K, N, Q, R)
Oxidation (M)
Water Loss (D, E, S, T)

After you press OK on the "Edit Structural Modification" and the "Edit Structural Modifications" dialog, you should check the checkbox in the "Structural Modifications" listbox next to the all of those new modifications.

Once you have told Skyline about those modifications it will reduce the number of warnings that you get about unidentified transitions.

Unfortunately, I see that there are still some more peptides that Skyline cannot figure out.
One problem is that you have some peptides that are supposed to have both the "Acetyl (N-term)" and the "Oxidation (M)" on the first amino acid. Skyline does not like to apply to modifications to the same amino acid, so in cases like this you usually have to define a single modification whose chemical formula is the combination of both of the modifications.

I have attached the document that I made with all of the necessary modifications. If you open the attached Skyline document and use "File > Import > Assay Library" it should succeed without any warnings.

If you would like to learn more about modifications in Skyline you might want to take a look at this webinar:

-- Nick