Hi Daniela,
You will need to provide some screenshots of your problem. You can use Alt-PrtScn to take them, paste them into a PowerPoint slide deck, and then attach them to a response to this thread.

It is also a good idea to use the Spectral Library Explorer to look at what got stored in the spectral library you created from your final_fragment.csv. You should be able to double-check the retention times for your MS/MS spectra in this interface to make sure they are what you expect, as explained in the MS1 Full-Scan Filtering tutorial in the "Verifying Library Retention Time Information" section:

https://skyline.gs.washington.edu/labkey/_webdav/home/software/Skyline/%40files/tutorials/MS1Filtering-2_5.pdf#page=11

The tip on "ID Annotations with Mascot Search Results" may also help to clarify things a little, despite the fact that you are not using Mascot search results:

https://skyline.gs.washington.edu/labkey/wiki/home/software/Skyline/page.view?name=mascot_missing_rt

Thanks for using Skyline. Happy to help more, if you can give me more detail (screenshots) of what you are seeing and what you are expecting.

--Brendan

Hi Brendan,

thank you for the information. Attached some screenshots. In the library are the correct RT, but in the peak area window not. I but three raw files from three different treatments. We already know that in A1 and A2 is close to nothing and in A3 should be something/much. Is it better to analyse each sample separate and compare later?

Thank you,
Daniela

I think I found an option. For the intense sample its now taking the correct RT.

Thank you,
Daniela

Hi Daniela,
The article on "ID Annotations with Mascot Search Results" is really applicable for you, and you should read it carefully:

https://skyline.gs.washington.edu/labkey/wiki/home/software/Skyline/page.view?name=mascot_missing_rt

Note that you do not have any ID annotations showing up in your chromatogram graphs. You should not continue processing this data until you do.

In this case, the problem is not with your RT values, but with the Source File values in your library. In the case you are showing in your screenshots, the spectrum source file is showing up as "Test2_IA_final_fragment.csv". With PLGS, Skyline should be able to remove the _IA_final_fragment.csv part, but then it will be looking for a raw data file named "Test2" to match with your spectrum source file. When you import a raw data file that does not match any of the spectrum source files in your library, then Skyline assumes you have no IDs for that raw data file, and this is pretty disastrous for MS1 peak picking.

True, you can improve matters with the option you have showing in your second set of screenshots, by limiting the range of retention time in which chromatograms are extracted (though 0.5 minutes seems a little narrow to me). In this case, if you have this option selected, but have not matching ID times in your file, Skyline will just use the ID times that do not match, assuming this is what you want, but it still won't use those times for peak picking (which we've actually improved in v2.6 to be released soon).

You will get the best peak picking if you go through the effort to make sure you spectrum source file names match your raw data file names, so that Skyline knows which raw data files your various IDs occur in.

Consult the tip on ID annotations carefully, and keep working until the ID annotations show up in your chromatograms. Then, reimport your data, and you should be much happier with the results.

Hope this helps. Sorry it is so complicated.

--Brendan

Thank you so much for your help, this somehow I missed. Now I have the ID, but still the fragment_final.csv term. It is also much better, samples where the peptide is not present does not come up with an ID. I think I can use even if he is keeping the fragment_final.csv, or?

Daniela

Hi Daniela,
Yes, this is fine. Looks good. I think you are on your way. Good luck with your research.

--Brendan