Problem importing modified peptide into library

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Problem importing modified peptide into library ronen gabizon  2018-11-28
 

Hello

We are analyzing samples of recombinant protein which were incubated with small molecules that attach covalently to cysteine. The objective is to identify the positions of modification and extract the chromatograms of the labeled peptides for characterization. Essentially I define the molecule's formula in MaxQuant (saved in the modifications.xml file) and define both the molecule and carbamidomethyl as variable modifications. The analysis by MaxQuant clearly identifies peptides containing the molecule as well as their sequence and the exact labeling site (including if there are two cysteines in the same peptide). My objective was to import the peptide search into skyline to build a spectra library and to extract the chromatograms of the carbamidomethyl and molecule-bound peptides. However, the peptides with the bound molecule are never added to the library - only carbamidomethyl peptides are. This is even though MaxQuant clearly identifies the modified peptides and they are defined in the same way.

Is there anything you can recommend to help in this problem?

Details: Windows server R2 enterprise 64 bit

        Skyline 64 bit 4.2.0.18305

         MaxQuant 1.6.0.16

I attach the screenshot from skyline. Cysteine containing peptides are added to the library (carbamidomethyl containing) and some are not (molecule-labeled).

I also attach the screen shot from the MaxQuant output showing the identification of the molecule-labeled peptide.

Thank you

 
 
Brendan MacLean responded:  2018-11-28

Hi Ronen,
This is the second time this has been reported in as many weeks. I hope we can fix the Skyline library builder soon to display an error message in this case instead of silently discarding the matches it doesn't understand, however, the fix is to put the modifications.xml or modifcations.local.xml, wherever the modification is defined into the same folder as your msms.txt.

That should be enough for the library builder to find your definition and add the modified forms to the library. Without that, Skyline release on a default modifications.xml file installed beside the Skyline.exe file, and it seems, silently discards any match with a modification that is not explained by that file.

Sorry for this silent failure issue. Always a bad idea to silently produce something that looks like what you asked for but is not really. Lots more of everyone's time gets wasted on this type of issue than more aggressively failing and then working on tuning the failures to allow things people really want to achieve.

Thanks for reporting this. Let us know if this suggestion fixes the issue for you.

--Brendan

 
ronen gabizon responded:  2018-11-28

Thank you. Unfortunately the peptide is still not added to the library after this step. Is there anything else you can suggest?

Ronen

 
Brendan MacLean responded:  2018-11-29

Hi Ronen,
Can you put your msms.txt, modifications.xml (optionally modifications.local.xml if you have one) and mqpar.xml into a ZIP file and post it to our support files drop point:

http://skyline.ms/files.url

If so, we can have a look and, I am sure, get to the bottom of it quickly.

--Brendan

 
ronen gabizon responded:  2018-11-29

Hi

Thanks, I uploaded the file. The modifications I was interested in are RA6 (C) (molecule that binds to cysteine residues) and Carbamidomethyl (C).

Ronen

 
matt.chambers42 responded:  2018-12-03

Hi Ronen,

The modifications.xml file you provided does not have the RA6 mod in it.

I've made a code change (which is not available in a -daily release yet) to make these missing mods an error instead of a warning, and also made the error a little more helpful; now it'll tell you that you've probably given it the wrong modifications.xml and/or modifications.local.xml file.