How do I include quantification for modifications not present in the sample?

How do I include quantification for modifications not present in the sample? kelvin luther  2023-03-29

I hope I can explain clearly what I would like to do. We work with small protein domains called EGFs, each of which could potentially be modified with as many as four EGF specific O-glycans. There is also non-stoichiometric hydroxylation of aspartates and asparagines. Additionally, mucin type O-glycans are possible in some cases, and we also have the potential for N-glycans. As you can imagine, if one is very unlucky, their peptide of interest could have a dozen or more potential glycoforms, in addition to the unmodified form. These modifications are often not stoichiometric, so we could potentially see all glycoforms in a given sample. When I make an extracted ion chromatogram (EIC) in Qualbrowser, I have masses for my peptide for all glycoforms. We use the observed values +/- a chosen ppm error for those we get hits for, and a calculated range for those we didn't get hits for. This allows us to plot an EIC that will show, from the raw data, that indeed, there is no peptide present for glycoforms that the software didn't identify. I am struggling to replicate this procedure in skyline. Firstly, it would be very advantageous to us if we had the ability to import a file of set modifications during each analysis, rather than having to imput them manually, or query a long list each time to make sure everything of consequence is selected. Secondly, I don't know how to get a curve in skyline for non-hits. We would very much like to query what came off the column for the non-observed glycoforms to show that they are not present in our data. I would much appreciate if anyone could give me some direction on how, or if this is possible in skyline. Thank you for your time.

Nick Shulman responded:  2023-03-29
I am not sure I understand your question.

One thing that Skyline is not good at is dealing with peptides that could not be found in the sample. Skyline always tries to choose the best peak, and so, if the peptide could not be found in the sample then Skyline ends up telling you about some peak areas that Skyline found somewhere else.
Unfortunately, we do not have a good answer for what to do about this yet. We do sometimes point people to the advanced peak picking tutorial, but that probably won't help in your case:

It sounds like you are asking about something else, but I do not understand what you are asking.
Could you send us your Skyline document?
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.

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

It might also be helpful if you could send us a screenshot of what you are asking about.

In terms of adding modifications to the document, there really is no easier way than doing "Settings > Peptide Settings > Modifications > Edit List > Add". The idea is that you would define all of the modifications that you need, check the checkbox next to all of those modifications in the list boxes at "Settings > Peptide Settings > Modifications" and then save your blank document. Then, whenever you want to create a new document you would copy that blank document and start from there.
If you needed to define a really large number of modifications, you could theoretically use a text editor on the ".sky" file. The Skyline document is an XML file and the syntax for how modifications are declared in the .sky file is pretty straightforward.
-- Nick