Can you send us your Skyline document and your .raw file?
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.

The Share Document dialog also gives you the option to include the raw files in the .zip, or you can send those to us separately.

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

Hello

Sorry for the late response.
Please find attached in the uploaded files (https://skyline.ms/files.url ) under the folder name: "envoi skyline support": the skyline document, the raw file and the fasta file used for the processing.
A proteomic platform processed a while ago our sample and gave us a list of 100 peptides including PTMs (Hydroxylation of lysine and proline).
So we expected a similar result using skyline, but unfortunately we dont. We would like to know the problem so we can test other new samples.

Thank you for your help

Skyline might not be the best tool for looking at DDA data like this.
Skyline is primarily used for quantifying samples based on chromatogram areas.

You can do a peptide search on the DDA data and then extract MS1 chromatograms, but that's usually not the right thing to do if you are interested in looking at modified peptides because their abundance is usually insufficient to be quantified using MS1 chromatograms.

When you did your peptide search in Skyline, which FASTA file did you use? The FASTA file that you uploaded had only one protein in it. You would never be able to find any peptides with a FASTA file that small because part of the peptide search involves searching for decoy (usually reversed) peptides based on the FASTA provided, and the true matches are deemed to be those that are better than nearly all of the decoy matches.
When I searched your .raw file using a FASTA file for the entire human proteome I did manage to find a lot of peptides including several from the Collagen protein in your original FASTA file.
-- Nick

Hello
Thank you for your reply. We have opened your document and we can find a dozen of peptides related to our protein of interest. You are right, we previously used a fasta file containing only one protein. It may explain in part why the processing did not function. We saw that you added "human" library in the peptide settings, which we did not neither.
1- Do we need a spectral library for peptide identification in DDA analysis? How did you get the library? is it built following the bibliospec link on the skyline home page?

2-We tried to search for oxidations in your skyline document. We know based on our platform analysis that the oxidized peptides are very intense in the sample. Unfortunaltely no modifications were visible within the peptides listed in the skyline document. Is there anyway we can search for PTMs in our DDA data?

3-We want ultimately to identify peptides and their PTMs using DDA analysis without any knowledge of the enzymes present in the sample. Is this possible using skyline?

Thank you a lot for your help

The spectral library was created from the peptide search results.
That is, I did "File > Search > Run Peptide Search", told it to do a peptide search using MS Amanda, and at the end of the process that file "human.blib" was created. The reason it was called "human.blib" is that the name of the Skyline document was "human.sky".

Whenever you import peptide search results into Skyline, a .blib file gets created.
The reason for this is that BiblioSpec knows how to create a spectral library from peptide search results, so Skyline only needs to know how to read .blib files in order to support many types of search results.
You can see the full list of peptide search results that BiblioSpec understands here:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild

If you tell Skyline about your modifications before you do the peptide search, the search engine will look for those modifications.
You can define the modifications on the "Modification" tab at "Settings > Peptide Settings".
You can learn more about modifications in Skyline in this webinar:
https://skyline.ms/Webinar10.url

There is no way to tell Skyline that you have no enzyme. You might need to do your own peptide search outside of Skyline if you want to do an enzyme-less search.
-- Nick