PTMProphet MSFragger modification mass discrepancy

[Alex Zelter]

I am running PTMProphet (TPP v5.2.1-dev Flammagenitus, Build 202003100907-8034) on MSFragger (MSFragger version MSFragger-2.3) output and ending up with strange results in specific cases.

For example, for a specific PSM, 52782, MSFragger outputs:

<spectrum_query start_scan="52782" uncalibrated_precursor_neutral_mass="2288.8486" assumed_charge="3" spectrum="QEP2_2018_0812_AZ_033_az736_AZ.52782.52782.3" end_scan="52782" index="41840" precursor_neutral_mass="2288.8384" retention_time_sec="6104.521">

<search_result>

<search_hit peptide="RYKAAFTECCQAADK" massdiff="471.0195" calc_neutral_pep_mass="1817.8188" peptide_next_aa="A" num_missed_cleavages="2" num_tol_term="2" num_tot_proteins="5" tot_num_ions="56" hit_rank="1" num_matched_ions="23" protein="sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2" peptide_prev_aa="K" is_rejected="0">

<modification_info>

<mod_aminoacid_mass mass="160.0307" position="9"/>

<mod_aminoacid_mass mass="160.0307" position="10"/>

</modification_info>

<search_score name="hyperscore" value="44.421"/>

<search_score name="nextscore" value="12.357"/>

<search_score name="expect" value="4.415e-15"/>

</search_hit>

</search_result>

</spectrum_query>

This would indicate that C9 is +57, C10 is +57 and there is an additional unlocalized mass diff of +471.

After running TPP I end up with output like this in interact.ipro.pep.xml for that scan:

<spectrum_query start_scan="52782" uncalibrated_precursor_neutral_mass="2288.8486" assumed_charge="3" spectrum="QEP2_2018_0812_AZ_033_az736_AZ.52782.52782.3" end_scan="52782" index="36796" precursor_neutral_mass="2288.8384" retention_time_sec="6104.521">

<search_result>

<search_hit peptide="RYKAAFTECCQAADK" massdiff="471.0195" calc_neutral_pep_mass="1817.8188" peptide_next_aa="A" num_missed_cleavages="1" num_tol_term="2" num_tot_proteins="5" tot_num_ions="56" hit_rank="1" num_matched_ions="23" protein="sp|P02768|ALBU_HUMAN" peptide_prev_aa="K" is_rejected="0" protein_descr="Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2">

<modification_info modified_peptide="RYKAAF[618]TEC[160]C[160]QAADK">

<mod_aminoacid_mass position="6" mass="618.0879"/>

<mod_aminoacid_mass position="9" mass="574.0287"/>

<mod_aminoacid_mass position="10" mass="574.0287"/>

</modification_info>.......

So it seems to me that we now have a modification mass addition of +471 on residues 6 9 and 10 rather than a mass addition of +471 on residue 6 and then an addition of +57 on residues 9 and 10.

In cases where there is just a mass diff (open modification) and no defined variable modifications output is as expected. This situation seems to occur when both defined variable modifications and open modifications are present.

I have confirmed the same behavior in older versions of MSFragger and TPP.

Any ideas would be much appreciated!

Thanks,

Alex

[David Shteynberg]

Hi Alex,

This seems like a bug that should be fixed.  Are you running PTMProphet at any point in your analysis pipeline?

Thanks,

-David

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[Alex Zelter]

Hi David,

Thanks for the fast response!

My list of commands are as follows (after running MSFragger):

tpp/bin/InteractParser       interact.pep.xml FraggerOutput.pepXML

tpp/bin/DatabaseParser       interact.pep.xml

tpp/bin/RefreshParser        interact.pep.xml /path/to/database/fastaFile.fasta

tpp/bin/PeptideProphetParser interact.pep.xml ACCMASS DECOYPROBS DECOY=random NONPARAM MASSWIDTH=520 MINPROB=0

tpp/bin/InterProphetParser   interact.pep.xml interact.ipro.pep.xml

tpp/bin/PTMProphetParser MASSDIFFMODE interact.pep.xml

tpp/bin/PTMProphetParser MASSDIFFMODE interact.ipro.pep.xml

I'm happy to share the full set of files with you if that helps.

Thanks!

Alex

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[David Shteynberg]

Thanks Alex.  I suspect it is a possible bug in PTMProphet. Can you see what step of your analysis created these additional mods on C?  I would help me narrow down the places to look.

Cheers,

David

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[Alex Zelter]

Hi David,

Your v5.2.1-dev Flammagenitus, Build 202003241419-80410 has fixed this problem.

Thank you!

Alex

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