Assistance Needed with Warning Messages in PTMProphet for TPP 6.3.3 PTM Localization

[Longping Fu]

I am currently working with PTMProphet in TPP version 6.3.3 for PTM localization. While PTMProphet successfully completes the task, I am encountering a significant number of warning messages in the console.

To provide some context, my workflow involves utilizing MSFragger as the search engine to convert raw files to mzML files and then searching against a database. For the modification parameters, I have set M 15.9949, N-term 42.1016, STC 299.123, and C 57.02146 as variable modifications, with no fixed modifications specified.

Upon obtaining the pepXML file from MSFragger, I employed Percolator for PSM validation using the following settings: --only-psms --no-terminate --post-processing-tdc. The resulting pepxml file was then processed with PTMProphet using the command: STC:299.123 MINPROB=0.5 MODPREC=0 FRAGPPMTOL=15 NOSTACK MAXTHREADS=1.

However, during the PTMProphet processing, I encountered warning messages similar to the following:

[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: S[386]CNGGSHPR
Neutral Mass (from pepXML) = 1254.52
BAD (SpectraST::Peptide) = S[386]CNGGSHPR
Neutral Computed Mass for Evaluation = 1212.51
PPM difference = 33487.5
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...
[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: SC[402]NGGSHPR
Neutral Mass (from pepXML) = 1254.52
BAD (SpectraST::Peptide) = SC[402]NGGSHPR
Neutral Computed Mass for Evaluation = 1212.51
PPM difference = 33487.5
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...
[WARNING:] Illegal PTM-shuffled peptide detected with non-matching mass: SCNGGS[386]HPR
Neutral Mass (from pepXML) = 1254.52
BAD (SpectraST::Peptide) = SCNGGS[386]HPR
Neutral Computed Mass for Evaluation = 1212.51
PPM difference = 33487.5
[INFO:] please check your specified modification masses for precision or adjust MODPREC= parameter ...

This pattern repeats for various peptides, indicating that PTMProphet is struggling to localize modifications on S, T, or C. The tool suggests adjusting parameters, specifically the MODPREC= parameter.

Has anyone encountered a similar issue with PTMProphet and successfully addressed it? I would greatly appreciate any insights, advice, or suggestions regarding this matter.

[David Shteynberg]

Dear Longping,

Maybe you can compress your analysis pepXML and mzML files and post them somewhere so I can replicate the issue locally?

Meanwhile, you can try the specifying all variable mods used in the search, when you run PTMProphet by changing the modification string as follows:

 M:15.9949,n:42.1016,STC:299.123,C:57.02146

Cheers!

-David

--
You received this message because you are subscribed to the Google Groups "spctools-discuss" group.
To unsubscribe from this group and stop receiving emails from it, send an email to spctools-discu...@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/ae1adab3-5501-4f29-b9d2-44d6252e7e84n%40googlegroups.com.

[Longping Fu]

Dear David,

I appreciate your swift response. I've compressed the pepXML and mzML files and uploaded them to Google Drive. You can access them through the following link: https://drive.google.com/file/d/1P463uxdvw67ZI7axiZO1OZOIlEZZg5qh/view?usp=sharing

Kindly confirm if you can successfully open the files.

Additionally, I'll be following your suggestion to include all variable modifications and check for any remaining warning messages.

Thank you for your assistance.

Sincerely,

Longping

[David Shteynberg]

Thanks Longping, I got your files.

To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/27485be2-f0b5-411e-a4e7-13cc49e588d5n%40googlegroups.com.

[Longping Fu]

Dear David, 

Thank you for providing your suggestions. I implemented your advice by including all variable modifications in the search, and the warning messages did not appear. 

I appreciate your assistance.

Sincerely,

Longping