Modifications for this peptide do not match current document settings

Modifications for this peptide do not match current document settings michael plank  2019-04-05


I built a library in Skyline from a .pep.xml I exported from Proteome Discoverer 2.0 using SequestHT as search engine. (The MS-run was on a standard phospho-peptide and I exported only a single PSM. I also exported the .mzML from the same PSM).
The library build succeeded, but when I go to View - Spectral libraries I get 'Modifications for this peptide do not match current document settings.' and it is complaining about a S[+79.9799] modification. (Due to this problem I cannot add the library spectrum to the document.)

The monoisotopic mass for phosphorylation is 79.966331 (79.9799 is the average mass). I checked that this is configured correctly in PD.
The .pep.xml has a line:
2. Dynamic Modification: (...) DeltaMass="79.96633" DeltaAverageMass="79.97990"

(This is on Skyline 64-bit

Can you please help me to get around this problem?


Nick Shulman responded:  2019-04-05
One thing that you could do is install Skyline version 3.7, and build your library using that older version of Skyline.
Skyline version 3.7 and earlier used only one decimal place to describe modifications in BiblioSpec library.
You can install older versions of Skyline from the "Unplugged" installer page here (click the "Archive" link):

Also, if you know how to run BlibBuild from the command line yourself, you can use a current version of BlibBuild, and omit the "-H" command-line argument that tells BlibBuild to use more than one decimal.

You can tell Skyline that you are interested in average masses instead of monoisotopic masses on the settings page:
Setting > Transition Settings > Prediction
I actually do not know whether that has any effect on the way that Skyline figures out what modification corresponds to the masses in a spectral library (I suspect that this setting will not fix your problem).

You could try sending us your peptide search results, and we might be able to figure out something else that would work.
If your files are less than 50MB, you can attach them to this support request.
Otherwise, you can upload them here:

-- Nick
michael plank responded:  2019-04-07
Hi Nick,

thanks a lot for the suggested workarounds. I`ll give it a try. It would still be nice if it were possible to work with the accurate mass as it seems to be present in the PD export. I`m attaching the .mzML and .pep.xml

Nick Shulman responded:  2019-04-07

Thanks for sending that simple .pep.xml file.
That file contains the line:
<mod_aminoacid_mass position="3" mass="167.01192835" />
BiblioSpec knows that Serine has the monoisotopic mass of 87.032028435, and therefore subtracts that number from the modified amino acid mass and comes up with 79.97989992, and so that's what BiblioSpec thinks the modification's mass is.

That number 167.01192835 does not make sense as either a monoisotopic mass or an average mass, since it's what you get from adding the monoisotopic mass of Serine (87.032028435) to the average mass of a Phosphate modification (79.9798979947423).

I am not sure which piece of software is responsible for deciding how to represent that modification in the .pep.xml file. It might be that you could work around this issue by lying to Sequest and saying that the DeltaMass and DeltaAverageMass for that modification are both 79.96633. I believe Sequest does not care about the DeltaAverageMass since you have specified "Use Average Precursor Mass: False" and "Use Average Fragment Mass: False" but maybe it still has some effect on the generation of the .pep.xml file.

Maybe someone else on this support board is more of an expert on Sequest HT or Proteome Discoverer and knows what to do about this.
-- Nick
engj responded:  2019-04-07
Brendan asked me to take a look at this thread and see if I had any insights. There's nothing else I can add beyond suggesting that the Proteome Discoverer's pep.xml file export has a bug. I'll forward this issue to a contact within Thermo to hopefully address the issue.

Given that a fix from Thermo is likely a long time out, I would suggest a short term fix would be to modify the exported pep.xml file before library building. i would issue a global find-and-replace of the text <u>mass="167.01192835"</u> to <u>mass="166.998359"</u> which would address phospho serine masses. Make the corresponding substitutions for the threonine and tyrosine phospho modification masses as well. Substitute whatever is reported for threonine to <u>mass="181.014010"</u> and tyrosine to <u>mass="243.029660"</u>. Note that I don't know if this will work as I haven't tried it myself but that's what I would try.

michael plank responded:  2019-04-08
once again, the support for Skyline is awesome. Thanks a lot!

I just set up a new phosphorylation definition in PD where I entered the monoisotopic mass also in the average mass field. When I use this for the search the library build works!
So it`s probably really a PD bug.

Thanks again,
engj responded:  2019-04-10
To give some closure to this thread, a SW Engineer at Thermo says that this bug was present in versions up to Proteome Discoverer 2.1 and was fixed in Proteome Discoverer 2.2.