Issue: "The following modifications could not be interpreted"

Issue: "The following modifications could not be interpreted" Mark Athanason  2020-06-29

Hello Brendan,

I saw another post on your forums from someone with a similar issue, but mine is different. Long story short I performed a search using Comet with a few variable modifications on K,R,S,T,C (drug adducts) along with oxidation of M. When I import my data into skyline to build a spectral library (using the wizard) the Add Modifications window lets me pick one of my adducts and the oxidative M. These are rounded to the nearest one's place. The modifications that cannot be interpreted have 2, 4, and 5 decimal places. In my comet parameters file I have the mass going out to 4 or five decimal places for all variable mods. If you can let me know how to circumvent this using the search data rather than manually entering the variable mod in skyline (future up-scaling of the project), I would appreciate it.


Brendan MacLean responded:  2020-06-29
Title: Issue: The following modifications could not be interpreted

Hi Mark,
Skyline only needs you to manually enter a definition for your modifications once. If Skyline is not recognizing them, then that means that they are not defined in Unimod, or defined more recently than our last update. Do you feel they are represented in Unimod?

How extensively do you generate new novel PTMs?

The benefit to having you fully defining a PTM is that this operation usually involves supplying a chemical formula for the PTM, where the best we could hope to do from most search engine output is a numeric delta-mass. Numeric detlas negatively impact the ability to generate accurate isotope distributions, and more generally, Skyline no longer knows the full atomic composition of the molecules being targeted.

So, while we could add a work-around to automatically generate mass-only PTMs from most spectral libraries, we would need convincing of its necessity for an important workflow. Currently our expectation is that there should be relatively few modifications no in Unimod, and users that it is not a great burden for users to fully define the ones they want to use.

If you still feel this is not the case with you, please explain further. Maybe provide the modifications and explain why they would define a large enough space to warrant automating mass-only modifications without chemical formulas. Alternatively, we can explain to you how to create a document with extensive PTM definitions which you can share across Skyline instances, which has worked for other applications as extensive as glycopeptide modifications.

Thanks for posting your case to the Skyline support board.


Mark Athanason responded:  2020-06-29

Hi Brendan,

Thanks so much for the response! For now I will add the empirical chemical formula for my protein adducts in skyline as variable mods. In the future, there will be a lot, but the project is in its infancy still.

Just to clarify, I can add the variable modifications to skyline prior to importing the data, and then they will not appear under the "The following modifications could not be interpreted" dialogue (if they are in fact found in my search)?

All the best,

Brendan MacLean responded:  2020-06-29

That is correct. If you have appropriately defined modifications available under Peptide Settings - Modifications - Structural Modifications, then Skyline should automatically find and use them for your library. They do not even need to be checked in that list. Skyline will automatically handle checking them when that is necessary.

You can also start with a blank document, add all of the modifications you every expect to use (as others have done for glycopeptides), check them all in the blank document, and save that document with some appropriate name, e.g. Then, anyone on your team that wants to get started using those modifications, without requiring a more complete document which actually uses them on targets, can simply open that template document once and close it again. Skyline will populate the modifications list with all the custom modifications in that document.

This is a great way to share custom modifications which do not appear in Unimod. Anyone who opens the modifications template document essentially extends the set of modifications which Skyline know about.

Hope this helps. Our collaborators monitoring glycopeptides were pretty happy once they understood how this works.