There have been a few support requests where users have had similar errors:
https://skyline.ms/announcements/home/support/thread.view?rowId=64428
https://skyline.ms/announcements/home/support/thread.view?rowId=54694
https://skyline.ms/announcements/home/support/thread.view?rowId=54050

I believe when you get an error like that it means that BiblioSpec is using the wrong .tsv file to go with the .speclib file.
If you send us all of your files we could try to take a look at it and figure out what is going on.
You package everything in a .zip file and upload it here:
https://skyline.ms/files.url

-- Nick

Hi NIck,

Thanks so much for sharing these similar issues.

Now I am pretty sure that my imported library is NOT the one which was used to generated the report.

BW

Hi,

Now I tried a newer version of DIA-NN (version 1.9) and I got the correct spectral library for the report.tsv file. Everything works fine until it starts to import my chromatogram files (.raw files). Only a few raw files were sucessfully imported while most files indicate "Failed". The error information says "Several peptides are missing..", as attached below. I clicked "Retry" and then the software crashed unfortunately.

Do you know how to overcome this issue?

Thanks in advance!

Yuxing

Usually that error tells you that your Skyline document has an iRT calculator defined, but the document is missing some of the iRT standard peptides.
You can take a look at the peptides that are in your iRT calculator by going to the "Prediction" tab at "Settings > Peptide Settings" and pushing the calculator button and then "Edit Current".

However, considering the fact that you got the error when extracting chromatograms from some but not all of your data files, those iRT standard peptides must be in the document, but, for some reason, during chromatogram extraction, Skyline did not think they were there.
I am not sure exactly how that can happen.

It would be helpful if you could send me your Skyline document and a couple of your raw files. You could send me all of your raw files, or maybe just one .raw file which has an error and one that does not.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms and spectral libraries.

The Share Document dialog also gives you the option to include .raw files in the .zip file or you could send them to us separately.

Files which are less than 50MB can be attached to these support request.
You can always upload larger files here:
https://skyline.ms/files.url

By the way, if you would like you learn more about using iRT in Skyline, the iRT tutorial has a lot of good information:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_irt
-- Nick

The error message that Skyline was showing you was misleading.
The error said:
The calculator DPP9_WT_KO requires all 74 of its standard peptides to be in the targets list in order to determine a regression. The following 31 peptides are missing:

The error should have probably said:
The calculator DPP9_WT_KO requires that at least 59 of its 74 standard peptides to have been detected. The following 31 peptides were not detected:

In order for Skyline to use iRT, it needs to find at least 80% of the iRT standards.
-- Nick

Hi Nick,

Thanks so much for your answers! FYI, my raw files are just different fractions rather than biological replicates and many peptides are just found some fractions (NOT all fractions). I guess that could explain why many standard peptides were missing in some raw files.

To overcome this issue, I started a new DIA-NN session with only 1 or 2 fractions (raw files) and imported the report and library into the Skyline. Then it worked nicely!

Now the problem has been solved. Thanks again for your kind help!

BW
YX