That dialog does not appear to be saying that any of your peptides are getting dropped as "Unmapped".
It says that many proteins (not peptides) are not being included in your document because they have no matching peptides.
(I am pretty sure that the Associate Proteins dialog is never supposed to remove any peptides from your document).

When you use the Import Peptide Search wizard, it is roughly equivalent to doing the following steps individually:
1. Create a spectral library by pushing the "build" button at:
Settings > Peptide Settings > Library
2. Use the menu item "File > Import > FASTA"
3. Tell Skyline to extract chromatograms using the menu item "File > Import > Results"

You might want to cancel out of the Import Peptide Search wizard after it has created a spectral library from your peptide search results.
Then, instead of doing "File > Import > FASTA", you could go to:
View > Spectral Libraries
and push the "Add All" button.

The "Add All" button on the Spectral Library Explorer is sometimes a good way to make sure that all of the peptides in the spectral library are added regardless of whether Skyline thinks that those peptides can be found in the FASTA file, or there are other reasons that the peptides are not passing the filter criteria specified at "Settings > Peptide Settings > Filter" or "Settings > Transition Settings > Filter".

If the "Add All" button on the Spectral Library Explorer does not do what you want it to, then I probably have not understood your question. Could you send us your Skyline document and FASTA file.
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 and your FASTA file are less than 50MB you can attach them to this support request. You can upload larger files here:
https://skyline.ms/files.url
-- Nick

Dear Nick,

That is what I was looking for, thank you!

Best wishes,
Hanna

Dear Nick,

I am using the command line interface of Skyline for high-throughput data processing. Do you have an example of a command line realisation of this process?

Best wishes,
Hanna

What are you hoping to do with the command line in Skyline?
You can find documentation about all of the command line commands in Skyline with the menu item "Help > Documentation > Command Line"
I am not an expert on the Skyline command line, so I would be answering your question by searching through that documentation window. (someone else on this support board might be able to give you better answers)

I do not see any command line command for creating a spectral library from peptide search results (other than the "--import-search" commands). It might be that if you want to create a spectral library similar to what you can do with the "Build" button on "Settings > Peptide Settings > Library" your batch file would need to invoke BlibBuild.exe and BlibFilter.exe. Or, it might be that "--import-search" will do what you want.

By the way, do you know about "Skyline Batch"? That tool makes invoking the Skyline command line easier:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=skyline-batch

-- Nick

Dear Nick,

Thank you very much for the fast reply.
I managed to create a spectral library via command line using the BiblioSpec tool. As far as I can see I am now lacking the correct chromatogram loading flags. I tried to load all raw files via the `--import-all-files` or `--import-filename-pattern`. This does not seem to load chromatograms correctly. Do you know a better way?

Best wishes,
Hanna

Make sure that there is a "--save" at the end of all of the arguments that you are passing to SkylineRunner.exe or SkylineCmd.exe.
I believe that if your argument list does not include a "--save" at the end, Skyline will do all of the work of extracting chromatograms, but then throw all that information away because it was never told to save it.

If that does not solve your problem, then you should give us more information about what you are telling Skyline to do and what the output looks like.
-- Nick

Dear Nick,

I think that did the job, thank you!

Best wishes,
Hanna

Out of curiosity: you said earlier that "many proteins (not peptides) are not being included in your document because they have no matching peptides". Does this mean that Skyline cannot identify any peak in the given raw file for the given scan number?

BW,
Hanna

I meant that the peptide was not identified in the peptide search results.
I am not sure whether you were importing a peptide search using search results which you had made outside of Skyline, or whether Skyline had performed the peptide search using MSAmanda, MSFragger, or MSGF+. Either way, I think that message was telling you that there were some proteins in the FASTA file for which no peptides had been detected in the peptide search results, and so those proteins would not be added to your Skyline document.
-- Nick

Hi,
I performed a search outside Skyline and imported the search results as .ssl file. I just double-checked, all the sequences in the fasta document are definitely in the ssl file. I still get the message that some peptides are unmapped.

Can you send me your .ssl file, fasta file and 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, the .ssl file and the fasta file are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url

-- Nick

Hi Nick,

Unfortunately I cannot share my data.
I noticed that all the unmapped peptides contain cysteine residues, whereas the mapped ones do not contain any cysteines. We did not introduce any cysteine modifications in the experiment, if at all, it could be thinkable that for some peptides the cysteine oxidation state changes. But this would not affect all peptides containing cysteines. Do you have an explanation why peptides containing cysteines might not be found? These sequences are definitely in the .ssl file.

Best wishes,
Hanna

When you go to:
Settings > Peptide Settings > Modifications
is there a checkmark next a Carbamidomethyl modification?

By default the Skyline document has Carbamidomethyl as an implicit modification which means that Skyline will apply that modification to all Cysteines unless you right-click on the peptide in the Targets tree and choose "Modify" and remove the modification.

If you would like to learn more about explicit and implicit modifications in Skyline you should look at this webinar:
http://skyline.ms/webinar10.url

If there are some peptides which are in your library which are not in your document, you can find them by going to:
View > Spectral Libraries

The spectral library (.blib file) only contains modifications masses. Skyline always has to figure out what the names of the modifications are that explain those masses.
If Skyline cannot figure out what modifications are on a particular peptide in the spectral library, the peptide sequence will be missing the icon next to it in the Spectral Library Explorer.
If you hover over the peptide sequence in the Spectral Library Explorer, the tooltip might give you a hint about why Skyline could not explain the modifications on that peptide.

I think if you have a checkmark next to the Carbamidomethyl modification in the peptide settings, Skyline will not let you add any peptide sequence with unmodified cysteines to the document. You will probably be able to fix this problem by going to:
Settings > Peptide Settings > Modifications
and unchecking the checkbox next to Carbamidomethyl.
-- Nick

Dear Nick,

That worked, thank you!
Just out of curiosity: did this functionality of Skyline assuming fixed Cys-CAM change between versions 21.2 and 22.2?

Best wishes,
Hanna

The behavior of this Carbamidomethyl modification has not changed in a very long while. In a clean installation of Skyline, the Carbamidomethyl is selected by default.
Skyline remembers the settings of the last document that you had opened.
When you create a new document, its settings end up being the same as the last document you had looked at (even if you have exited Skyline in between).
If you are going to be working with documents which have different settings, we recommend that you create blank documents that have the appropriate settings in them. When you want to create a new document, instead of using the "New Document" menu item, we recommend that you open up one of those documents that you already have which has the correct settings, and do "Save As" to save it to a new place where you can work on it. (That is, we recommend that you have "Template Documents" with the correct settings in them).

If you want to restore all of Skyline's settings back to their defaults, there's a "Clear All Saved Settings" button at:
Tools > Options > Miscellaneous

-- Nick