My DDA data file was acquired on Orbitrap, in *.raw format. Is this the reason why it can't be recognized? Thanks,

Can you provide some screenshots so we can better understand exactly what you're seeing?

Do you have the .raw file that was searched by Mascot in the same directory as the Mascot result file?

Thanks for using the Skyline support board,

Brian

Thanks a lot, Brian. I added .raw, .mzid, .dat, .mgf all in the same directory and finally can find the result file. Now I go through this step. I can see the integration, but new problem is only a small part of peptide identifications were loaded into Skyline. I have many modifications, very unique, like a 301 mass added to K, W, and N-term and 167 mass added to K, W, and N-term. The majority of such identification were lost. I should have 2370 peptides, but now only a few hundreds are shown.

When you look in the spectral library that was created during the peptide search import, do you see the peptides and modifications you expect? If so, then it's probably something in your settings that's filtering things out. If not, there may be an issue with the import step, possibly with your more exotic modifications (but that's just a guess).

During the peptide search import, I saw some modifications, but not the ones I expect. It listed some modifications that couldn't be interpreted. I tried to add modifications, but it seems new modifications can't be displayed during peptide search import and in final integration step. Is there any way to add special modifications and make them appear during the peptide search import, please?

In Settings: Peptide Settings, I already added "Analyte (KW)" as a new modification, but it can't be accepted by Skyline. Is there any other way to edit the modification list in Skyline, please? Thanks a lot

>> It listed some modifications that couldn't be interpreted.

That is certainly the issue, then. There is some problem understanding the modification descriptions.

What we want, of course, is for this to just work when you do the import. Can we have a look at the .raw, the Mascot files, and the Skyline document you were using when you started the import process?

Please see the Skyline Support page (https://skyline.ms/project/home/support/begin.view?) for information on uploading all that. Ideally in one big zip file.

Thank you so much for the helps. I attach the zip file as requested.

Thanks - we'll need the files you started with, as well: the Mascot files and the .raw files.

.dat

.mgf

.mzid

.raw is still too big to be uploaded since its size is ~500 MB after zip. That will be great if you suggest one way to send you the file. Thank you really much,

Please use our file upload page:

http://skyline.ms/files.url

Thanks

Thanks, F002498 raw.zip has been uploaded.

Identifications may be missing from your spectral library for two reasons:
1) they are being excluded due to the cut-off score. You can lower the cut-off score in order to include identifications with poorer scores.
2) when Skyline builds spectral libraries, if there are multiple identifications for the same spectrum, all of them are discarded. You can check the "Include ambiguous matches" option to prevent this behavior.

If your spectral library contains modifications that Skyline doesn't have information about, you will need to add them manually. You can add modifications to Skyline in several places:
1) during Import Peptide Search on the "Add Modifications" page, click the "Add modification..." button near the bottom.
2) in Settings > Peptide Settings > Modifications, click the "Edit list..." button, then "Add...".
3) when opening a spectral library in the Spectral Library Explorer (View > Spectral Libraries), click the "Add to Document..." button near the bottom of the "Add Modifications" dialog that appears.

Thanks,
Kaipo

The modifications were added on the “add modifications “ page, during Import Peptide Search,and Peptide Settings, Edit list as said in 1) and 2). These two were done both. But in “Extract Chromatogram”, there were these modifications that can be selected and added into document. I will see the effect to lower cut-off score, hope this can work. Can you extract about 30 different K sites with the modifications using my uploaded files? Thank you very much for the advices,

Even in blank project，this modification was added in Peptide Settings ，and selected together with carbamyl，deamidation。Then input a Peptide sequence manually ，one peptide will transform to multiple peptides containing different modifications 一but the new modification I added wouldn’t be included. It seems this modification can’t be recognized by Skyline even though it has been added in Peptide Settings and checked. Did you see this before?

I am not sure that I understand your question. Can you post a screenshot of what you are seeing?