If you want to analyze fragments which are not attached to any amino acids from the peptide sequence, then you should add them to the "Special Ions" list on the "Filter" tab at "Settings > Transition Settings".

Once you have defined the special ions in the Transition Settings, you can add them to Targets in the Targets tree by right-clicking on a Precursor and choosing "Pick Children".

If, instead, you wanted to monitor the part of the molecule that remained after the special ion fell off, then that would be a "Neutral Loss" which you would specify in the Modification definition.
But, fragments which are not attached to the peptide are "Special Ions".
-- Nick

Hi Manousos,

Attached you will find a Skyline template that might get you started. Also some screenshots to illustrate what Nick described. Keep in mind that for the special ions if you leave them "grey" these will only be used to annotate the fragment ion spectra. If you leave them ticked black then it will also generate "transition" targets.

Hope this helps and good luck glycohunting!

Sincerely,
Juan C.

Dear Nick, Dear Juan,

Thanks a lot for your help and prompt replies. This was very helpful! Dear Juan, it seems I have a problem openning the skyline template you kindly sent. I have the 24.1.0.414 skyline version installed and when I try to open the file I receive a failure message because the document format version is 24.11 which is newer than the 24.1. I searched for a more updated version, but 24.1 is the best I can find. Can you please help?

Thanks a lot in advance for your time and consideration.

Kind regards,
Manousos

That document can only be opened by Skyline-daily.
You can install Skyline-daily by clicking on the "Skyline-daily (beta)" button on the main Skyline page:
https://skyline.ms/project/home/software/Skyline/begin.view
If you are using Skyline-daily (or any version of Skyline) and want to produce a version of the document that can be opened by older versions of Skyline you can use the "File > Share" menu item and choose to save in an older version's format.
--Nick

Hi Manousos,

Attached a version saved in Skyline 24.1 using Nick's instructions.

Hope it works!

Sincerely,
Juan C.

Dear Nick, Dear Juan,

Thanks a lot for your prompt replies. This was really helpful!

Best regards,
Manousos

Dear Nick, Dear Juan,

I am trying to create the oxonium ion profile, but I am getting only the MS1 extraced ion chromatogram which is not helpful. I tried to use the molecules interface or the mixed one (molecules and peptides) since I am not really interested in specific peptides. What I need to do is to create oxonium ions profile for specific .raw files (un-digested samples) at the MS2 level. Practically, what I want to see in the end is the peaks (if any) of the oxonium ions (eg HexNAc, Hex, etc) in the MS/MS spectrum of a .raw file during time. I attach a doc file for your convenience which is from the study of Madsen et al. showing what I would like to do.

Thanks a lot in advance for your invaluable help and support.

Best regards,
Manousos

I do not understand your question but if you send us your Skyline document and your raw files we might be able to figure it out.

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, or you could send those to use 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

Dear Nick,

Please find attached the zip file kindly requested. A representative raw file has been submitted to the https://skyline.ms/files.url (raw file name: 200409L000008).

Thanks a lot,

Manousos

Thank you for uploading that Skyline document and .raw file.

Skyline is not the recommended tool for looking at DDA data.
In a DDA experiment, the mass spectrometer decides which m/z's to fragment based on what it sees in the MS1 spectra. Because of this, there is often a long gap between the times when a particular precursor m/z was selected for fragmentation, so MS2 chromatograms extracted from DDA data tend to have long straight lines connected the times where a matching MS2 spectrum was acquired.

I see that in your Skyline document your precursor has the m/z of exactly 1000?
Is that the correct m/z?
In order for a MS2 spectrum to contribute to the extracted MS2 chromatogram, the isolation m/z of the spectrum needs to be very close to the declared precursor m/z in the Skyline document. Your document does not have any MS2 chromatograms because there were no spectra that were close enough.

What were you hoping to do with your data? If you can get by with just analyzing MS1 chromatograms, then Skyline might work for you. I would recommend that you tell Skyline the exact chemical formula for your molecules instead of just the m/z. When Skyline knows the chemical formula of a molecule, Skyline will extract multiple precursor chromatograms for the isotope envelope which results in more confident identification and quantification.

If your DDA MS2 spectra are important to you, then there probably exists a different tool which would work better for you.
-- Nick

Dear Nick,

Thanks a lot for your help and the prompt reply. Well, I was hoping I could have oxonium ions profile in MS2 spectra from DDA data. Practically, I am not interested at all for the precursors, I just needed to have the fragment profile (oxonium ions) in the MS2 spectra. I see your point that skyline might not be the best choice for the MS2 spectra profiling and if there is no other way to do this with skyline I will consider different options. The precursor in the file I sent you has a random value, it could be practically any m/z. The thing is that I am not interested for a specific only precursors to see its fragments, but I would like to see the MS2 profile in total hoping to highlight the oxonium ions m/z.

Best regards,
Manousos

Hi,

What about making Skyline think the DDA data is wideband DIA data (e.g. MSE) with "All ions" in the DIA transition settings. Do you envision any issues with the data import in that way, Nick?

Sincerely,
Juan C.