A fixed and variable modification on the same amino acid

A fixed and variable modification on the same amino acid laura declerck  2016-07-13

I study modified histone peptides. Because of the derivatisations every N-terminal AA contains a propionylation on the N-terminus. This modification was put fixed in Mascot. Sometimes the N-terminal AA is a Lysine which almost always contains a PTM.
I know skyline cannot recognize 2 different modifications on the same AA, so i have build new modifications for all the PTMs with the mass of propionyl added. However, Skyline cannot recognize any of these self-made modifications.

I think this is because Mascot reports the fixed and the variable modifications as separate delta masses and not as one.

Do you know how I can ensure that these peptides still get into my library?

Kind regards,
Nick Shulman responded:  2016-07-14
I'll send you email with instructions on how to send us files so that we can take a look and figure out why Skyline is not matching these modifications.
laura corveleyn responded:  2020-11-30

This issue was elegantly fixed now over 4 years ago. However, we are bumping into yet another situation where the problem is coming back. More specifically, we now have other PTM's on our N-terminus for the first time (e.g. N-terminal Acetylation) and Skyline still adds the propionylation mass to the N-terminus, resulting in a 98.03678 mass shift which he does not recognize. In other words, while both N-term propionylation and acetylation are set as variable in the peptide settings, the DAT file from Mascot depicts the fixed N-term Propionylation and Skyline adds that to the delta mass.

How could this be fixed? Or can you think of a workaround?

Kind regards,

other Laura ;)
Nick Shulman responded:  2020-11-30

Skyline does not know how to put two modifications on the same residue, so it is always necessary to define a single modification that has the chemical formula of the two modifications combined together.

It sounds like you have already been doing this, so I cannot think of a reason that this would not be working for you.

It would be helpful if you could 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 spectral libraries. If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:

One thing that does not get included in the .sky.zip is the modifications that you have defined but are not yet using in your Skyline document (i.e. some of the unchecked modifications at "Settings > Peptide Settings > Modifications".) In order for me to see the same modification matching behavior in the spectral library viewer, I need to see those settings too. In order to save those settings, you have to first use the menu item "Settings > Save Current" and give your settings a name. Than, you need to go to "Settings > Share" and export those settings to a .skys file. Please send us that .skys file as well as your .sky.zip.

 -- Nick
laura corveleyn responded:  2020-11-30
Dear Nick,

Thank you so much for this quick response!! Just to be clear: the problem is that Skyline changes an N-acetyl into a mass of 98Da because Skyline adds an N-terminal propionylation, most probably because the Mascot DAT file has it set to fixed? We have set the N-terminal propionyl to variable in Skyline, but that does not seem to make a difference. In other words: that 98Da is not on the peptide N-terminus in real life (or in the Mascot result for that matter), it is being created by Skyline, so we do not want to create an atomic formula to explain it.

You can find the files in the attachment.

Thanks in advance!

matt.chambers42 responded:  2020-11-30
Hi Laura,

Could you share one of the Mascot DAT files as well? Thanks.
laura corveleyn responded:  2020-11-30
matt.chambers42 responded:  2020-12-02
Hi Laura,

Are you saying that the variable mod Acetyl on the N terminus is supposed to take the place of the fixed Propionyl mod? If so, I don't think that's how it works. Fixed mods are always there, whether there's a variable mod or not. If the Propionyl mod is supposed to be mutually exclusive with Aceytl, you need to configure them both as variable. In these results, Acetyl mods really do co-occur with the Propionyl mods. Whether those are real peptides or not (vs a misinterpretation of the real peptide) I don't know.
Brendan MacLean responded:  2020-12-02
In other words, your Mascot search results are not in agreement with what you are requesting of Skyline. It seems like you would need to rerun your Mascot search to achieve what you are asking for. It sounds like you have Skyline configured correctly with both acting as variable modifications, but you should not have specified the fixed modification in your Mascot search, if I am understanding correctly.
laura corveleyn responded:  2020-12-08

Yes, the variable Acetyl is supposed to take the place of the fixed Propionyl at the N-terminus. In our Mascot search, we also have the Propionyl on K set to fixed, however, it works perfectly to replace it with any variable mod in Skyline. So somehow, Skyline can change the fixed Propionyl on K to a variable Acetyl, but can't do this for the N-terminus and keeps adding the mass of the variable mod (acetyl) to the fixed mod (propionyl).
matt.chambers42 responded:  2020-12-14
On further inspection, it does appear that Mascot overrides fixed mods with variable mods. And the Mascot team confirmed that to me. Oddly, BiblioSpec was actually coded back in 2016, probably prompted by this support request, to allow Mascot's variable mods to override fixed mods on residues but NOT on termini. So that's why we're seeing +98 on termini only. A fix for this might not be in the next (imminent) Skyline-daily, but will be in the one after that.