For experiments on a triple quadrupole, the best tutorials are "Targeted Method Editing" and "Targeted Method Refinement":
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_edit
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_refine

Can you send us the transition list that you are trying to insert? We might be able to figure out what is wrong with it.

It would also be helpful if you could send us your Skyline document so that we can make sure that you told Skyline about your heavy isotope modifications correctly.
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.
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

If inserting a transition list does not work for you, you could also get the peptides into your document using the "Edit > Insert > Peptides" menu item.
After you have inserted the peptides into the document, you can right-click on each Peptide in the Targets tree and choose "Modify" to tell Skyline what modifications are on the heavy peptide. You can also right-click on the Peptide and choose "Pick Children" in order to tell Skyline which precursors. In the Skyline targets tree, a Precursor represents a particular charge state and isotope label type. You can right-click on a Precursor in the Targets tree and choose "Pick Children" to specify which Transitions should be monitored.

Most people do not worry about assigning proteins to peptides when inserting a transition list. Instead, after the peptides are in the document, they use the menu item "Refine > Associate Proteins" to give Skyline a FASTA file, and then Skyline will move the peptides around in the Targets tree so that they are grouped by the Protein they belong to.

Hope this helps,
-- Nick

Dear Nick,

Thank you for your quick reply.
I will be having a look at the tutorials you shared, my analysis was done on a SCIEX QTOF, so I am not sure I will be able to replicate but I will try.

I have attached the list of transitions in this reply and I have share the Skyline document on your repository udner the name "Import_peptide_SFV". It is quite heavy so if it is easier for you I can recreate the document but only have a couple of chromatogram imported.

I did try to insert peptides and it has worked where I can see the light and heavy peptide as standard. However, I can only see one m/z for each and that does not change even if I change the transition settings to have p, b and y ions. So in the pick children, I don't have any choice and for some peptides it can't find any precursors.

Thank you for your help,

Best wishes,
Cecile

In the child picker window that appears when you do "Pick Children", there is a filter button which looks like an inverted triangle.
When that button is selected, Skyline will only display the choices that satisfy the settings on the "Filter" tab of Transition Settings.
If you unselect that button, Skyline will show you the complete list of possible transitions.

If you are really seeing only one choice in that window, then it is likely you are choosing Precursors to add to the Peptide. You should choose the option with the correct precursor charge and isotope label type.
After you have added the Precursor to the Targets tree, you should be able to right-click on the Precursor, choose "Pick Children" and choose the transitions that you need.

If it's still not working for you, you should post a screenshot of what you are seeing.

I'll take a look at your transition list.
-- Nick

Thank you for attaching that Excel file.
The problem is that all of your peptide sequences have a non-breaking space character at the end of them.
This looks identical to a regular space, except that Skyline has no idea what to do with it.
This is the reason that you are getting "Invalid peptide sequence DYSLFSYATK  found" errors.
There really is an extra character right after the "K" which is causing problems.

If you get rid of that special character from everywhere in your transition list then things will work much better.
-- Nick

Dear Nick,

Thank you for your help, as you said I did not realise that there was spaces at the end of each word/number. I was able to successfully import the transition list.

It might not be something that you would be able to help with, but once the transition list was imported, I imported my results (MRM data with a method that contained the exact same transitions from the transition list). However, Skyline was not able to find anything, "chromatogram info unavailable" although I had very intense peak for my heavy peptides.

Thank you very much,

Best wishes,
Cecile

If you send us your Skyline document and your .wiff/.wiff2 and .wiff.scan files we could probably tell you why the chromatograms are not showing up.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including whatever chromatograms might have been extracted.

The Share Document dialog gives you the option to include in the .zip file the raw files from which the chromatograms were extracted. It would be good to choose that option because with Sciex files, you always need to include the .wiff.scan files along with the .wiff or .wiff2 files, and Skyline will take care of that if you tell it to put them in the .zip file.

If that .zip file is less than 50MB you can attach it to this support request.
You can always upload larger files here:
https://skyline.ms/files.url

The usual reason that chromatograms do not show up would be that there really are no chromatograms whose Q1 and Q3 values match the precursor and product m/z's in the Skyline document. Usually, I use a program called ProteoWizard SeeMS.exe to see what chromatograms are available in the .wiff file.
-- Nick