PTMProphet MSFragger modification missing mass

[Alex Zelter]

I am running into a situation where MSFragger returns a massdiff but PTMProphet seems to ignore it. No modification_info or mod_aminoacid_mass position is reported by PTMProphet for some results, while for others things work as expected.

Here are 2 PSMs as examples. I've included both MSFragger xml output and PTMProphet output. For scan number 49967 the open modification is localized and reported correctly by PTMProphet, but for scan 74042 that information is missing from the PTMProphet output, despite the MSFragger xml input being similar.

Both the examples below are taken from the same results files and were thus treated identically. TPP v5.2.1-dev Flammagenitus, Build 202003241419-8041 was used.

If you have any idea what might be causing this I'd be very grateful!

Thanks,

Alex

MSFragger pep.xml output for scan 49967 (works as expected):

<spectrum_query start_scan="49967" uncalibrated_precursor_neutral_mass="1847.0168" assumed_charge="3" spectrum="QEP2_2018_0812_AZ_033_az736_AZ.49967.49967.3" end_scan="49967" index="39256" precursor_neutral_mass="1847.0134" retention_time_sec="5830.280">

<search_result>

<search_hit peptide="VTKCCTESLVNR" massdiff="495.3547" calc_neutral_pep_mass="1351.6587" peptide_next_aa="R" num_missed_cleavages="1" num_tol_term="2" num_tot_proteins="10" tot_num_ions="44" hit_rank="1" num_matched_ions="9" protein="sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2" peptide_prev_aa="R" is_rejected="0">

<alternative_protein protein="tr|A0A087WWT3|A0A087WWT3_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|A0A0C4DGB6|A0A0C4DGB6_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|B7WNR0|B7WNR0_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|C9JKR2|C9JKR2_HUMAN Isoform of P02768, Albumin, isoform CRA_k OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|D6RHD5|D6RHD5_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|H0YA55|H0YA55_HUMAN Isoform of P02768, Serum albumin (Fragment) OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="sp|P02768-2|ALBU_HUMAN Isoform of P02768, Isoform 2 of Serum albumin OS=Homo sapiens GN=ALB" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="sp|P02768-3|ALBU_HUMAN Isoform of P02768, Isoform 3 of Serum albumin OS=Homo sapiens GN=ALB" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="sp|ALBU_BOVIN|" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<search_score name="hyperscore" value="15.283"/>

<search_score name="nextscore" value="12.987"/>

<search_score name="expect" value="1.670e-04"/>

</search_hit>

</search_result>

</spectrum_query>

PTMProphet output for the scan 49967:

<spectrum_query start_scan="49967" uncalibrated_precursor_neutral_mass="1847.0168" assumed_charge="3" spectrum="QEP2_2018_0812_AZ_033_az736_AZ.49967.49967.3" end_scan="49967" index="34348" precursor_neutral_mass="1847.0134" retention_time_sec="5830.280">

<search_result>

<search_hit peptide="VTKCCTESLVNR" massdiff="495.3547" calc_neutral_pep_mass="1351.6587" peptide_next_aa="R" num_missed_cleavages="1" num_tol_term="2" num_tot_proteins="10" tot_num_ions="44" hit_rank="1" num_matched_ions="9" protein="sp|P02768|ALBU_HUMAN" peptide_prev_aa="R" is_rejected="0" protein_descr="Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2">

<alternative_protein protein="tr|A0A087WWT3|A0A087WWT3_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|A0A0C4DGB6|A0A0C4DGB6_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|B7WNR0|B7WNR0_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|C9JKR2|C9JKR2_HUMAN" protein_descr="Isoform of P02768, Albumin, isoform CRA_k OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|D6RHD5|D6RHD5_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|H0YA55|H0YA55_HUMAN" protein_descr="Isoform of P02768, Serum albumin (Fragment) OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="sp|P02768-2|ALBU_HUMAN" protein_descr="Isoform of P02768, Isoform 2 of Serum albumin OS=Homo sapiens GN=ALB" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="sp|P02768-3|ALBU_HUMAN" protein_descr="Isoform of P02768, Isoform 3 of Serum albumin OS=Homo sapiens GN=ALB" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="sp|ALBU_BOVIN|" num_tol_term="2" peptide_prev_aa="K" peptide_next_aa="R"/>

<search_score name="hyperscore" value="15.283"/>

<search_score name="nextscore" value="12.987"/>

<search_score name="expect" value="1.670e-04"/>

<analysis_result analysis="peptideprophet">

<peptideprophet_result probability="0.9495" all_ntt_prob="(0.0000,0.0020,0.9495)">

<search_score_summary>

<parameter name="fval" value="6.0889"/>

<parameter name="ntt" value="2"/>

<parameter name="nmc" value="1"/>

<parameter name="massd" value="-0.303"/>

<parameter name="isomassd" value="3"/>

</search_score_summary>

</peptideprophet_result>

</analysis_result>

<analysis_result analysis="interprophet">

<interprophet_result probability="0.919585" all_ntt_prob="(0,0.00121737,0.919585)">

<search_score_summary>

<parameter name="nrs" value="-8.8028"/>

<parameter name="nsi" value="0.999"/>

<parameter name="nsm" value="2.9825"/>

<parameter name="nsp" value="20"/>

</search_score_summary>

</interprophet_result>

</analysis_result>

<modification_info modified_peptide="VTK[623]CCTESLVNR">

<mod_aminoacid_mass position="3" mass="623.4497"/>

</modification_info>

<analysis_result analysis="ptmprophet">

<parameter name="mass_ppm_offset" value="-2.43401"/>

<parameter name="mass_ppm_tolerance" value="11.2026"/>

<ptmprophet_result ptm="MASSDIFF" prior="0.0833333" ptm_peptide="V(0.024)T(0.024)K(0.777)C(0.035)C(0.035)T(0.023)E(0.018)S(0.015)L(0.013)V(0.013)N(0.011)R(0.011)">

<parameter name="mean_best_prob" value="0.777"/>

<parameter name="norm_info_gain" value="0.580"/>

<parameter name="localized_mods" value="0.580"/>

<mod_aminoacid_probability position="1" probability="2.436e-02" oscore="0.071" mscore="0.250" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.500" nterm_score="0.000"/>

<mod_aminoacid_probability position="2" probability="2.436e-02" oscore="0.071" mscore="0.250" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.500" nterm_score="0.000"/>

<mod_aminoacid_probability position="3" probability="7.774e-01" oscore="0.928" mscore="0.667" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.500" nterm_score="0.000"/>

<mod_aminoacid_probability position="4" probability="3.531e-02" oscore="0.072" mscore="0.333" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.500" nterm_score="0.000"/>

<mod_aminoacid_probability position="5" probability="3.531e-02" oscore="0.072" mscore="0.333" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.500" nterm_score="0.000"/>

<mod_aminoacid_probability position="6" probability="2.256e-02" oscore="0.043" mscore="0.250" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.462" nterm_score="0.000"/>

<mod_aminoacid_probability position="7" probability="1.758e-02" oscore="0.033" mscore="0.200" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.417" nterm_score="0.000"/>

<mod_aminoacid_probability position="8" probability="1.491e-02" oscore="0.025" mscore="0.167" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.364" nterm_score="0.000"/>

<mod_aminoacid_probability position="9" probability="1.349e-02" oscore="0.024" mscore="0.143" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.300" nterm_score="0.000"/>

<mod_aminoacid_probability position="10" probability="1.255e-02" oscore="0.023" mscore="0.125" direct_oscore="0.500" direct_mscore="0.500" cterm_score="0.222" nterm_score="0.000"/>

<mod_aminoacid_probability position="11" probability="1.131e-02" oscore="0.019" mscore="0.100" direct_oscore="0.101" direct_mscore="0.333" cterm_score="0.000" nterm_score="0.000"/>

<mod_aminoacid_probability position="12" probability="1.082e-02" oscore="0.014" mscore="0.091" direct_oscore="0.101" direct_mscore="0.333" cterm_score="0.000" nterm_score="0.000"/>

</ptmprophet_result>

</analysis_result>

</search_hit>

</search_result>

</spectrum_query>

MSFragger pep.xml output for scan 74042 (does not work as expected):

<spectrum_query start_scan="74042" uncalibrated_precursor_neutral_mass="1494.4763" assumed_charge="2" spectrum="QEP2_2018_0812_AZ_033_az736_AZ.74042.74042.2" end_scan="74042" index="61738" precursor_neutral_mass="1494.4722" retention_time_sec="8409.789">

<search_result>

<search_hit peptide="VTKCCTESLVNR" massdiff="142.8135" calc_neutral_pep_mass="1351.6587" peptide_next_aa="R" num_missed_cleavages="1" num_tol_term="2" num_tot_proteins="10" tot_num_ions="22" hit_rank="1" num_matched_ions="11" protein="sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2" peptide_prev_aa="R" is_rejected="0">

<alternative_protein protein="tr|A0A087WWT3|A0A087WWT3_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|A0A0C4DGB6|A0A0C4DGB6_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|B7WNR0|B7WNR0_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|C9JKR2|C9JKR2_HUMAN Isoform of P02768, Albumin, isoform CRA_k OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|D6RHD5|D6RHD5_HUMAN Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="tr|H0YA55|H0YA55_HUMAN Isoform of P02768, Serum albumin (Fragment) OS=Homo sapiens GN=ALB PE=1 SV=1" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="sp|P02768-2|ALBU_HUMAN Isoform of P02768, Isoform 2 of Serum albumin OS=Homo sapiens GN=ALB" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="sp|P02768-3|ALBU_HUMAN Isoform of P02768, Isoform 3 of Serum albumin OS=Homo sapiens GN=ALB" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<alternative_protein protein="sp|ALBU_BOVIN|" peptide_prev_aa="R" peptide_next_aa="R" num_tol_term="2"/>

<search_score name="hyperscore" value="19.365"/>

<search_score name="nextscore" value="17.327"/>

<search_score name="expect" value="1.083e-05"/>

</search_hit>

</search_result>

</spectrum_query>

PTMProphet pep.xml output for scan 74042 (does not work as expected):

<spectrum_query start_scan="74042" uncalibrated_precursor_neutral_mass="1494.4763" assumed_charge="2" spectrum="QEP2_2018_0812_AZ_033_az736_AZ.74042.74042.2" end_scan="74042" index="54976" precursor_neutral_mass="1494.4722" retention_time_sec="8409.789">

<search_result>

<search_hit peptide="VTKCCTESLVNR" massdiff="142.8135" calc_neutral_pep_mass="1351.6587" peptide_next_aa="R" num_missed_cleavages="1" num_tol_term="2" num_tot_proteins="10" tot_num_ions="22" hit_rank="1" num_matched_ions="11" protein="sp|P02768|ALBU_HUMAN" peptide_prev_aa="R" is_rejected="0" protein_descr="Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2">

<alternative_protein protein="tr|A0A087WWT3|A0A087WWT3_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|A0A0C4DGB6|A0A0C4DGB6_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|B7WNR0|B7WNR0_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|C9JKR2|C9JKR2_HUMAN" protein_descr="Isoform of P02768, Albumin, isoform CRA_k OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|D6RHD5|D6RHD5_HUMAN" protein_descr="Isoform of P02768, Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="tr|H0YA55|H0YA55_HUMAN" protein_descr="Isoform of P02768, Serum albumin (Fragment) OS=Homo sapiens GN=ALB PE=1 SV=1" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="sp|P02768-2|ALBU_HUMAN" protein_descr="Isoform of P02768, Isoform 2 of Serum albumin OS=Homo sapiens GN=ALB" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="sp|P02768-3|ALBU_HUMAN" protein_descr="Isoform of P02768, Isoform 3 of Serum albumin OS=Homo sapiens GN=ALB" num_tol_term="2" peptide_prev_aa="R" peptide_next_aa="R"/>

<alternative_protein protein="sp|ALBU_BOVIN|" num_tol_term="2" peptide_prev_aa="K" peptide_next_aa="R"/>

<search_score name="hyperscore" value="19.365"/>

<search_score name="nextscore" value="17.327"/>

<search_score name="expect" value="1.083e-05"/>

<analysis_result analysis="peptideprophet">

<peptideprophet_result probability="0.9504" all_ntt_prob="(0.0000,0.9728,0.9504)">

<search_score_summary>

<parameter name="fval" value="6.1669"/>

<parameter name="ntt" value="2"/>

<parameter name="nmc" value="1"/>

<parameter name="massd" value="0.337"/>

<parameter name="isomassd" value="3"/>

</search_score_summary>

</peptideprophet_result>

</analysis_result>

<analysis_result analysis="interprophet">

<interprophet_result probability="0.865139" all_ntt_prob="(0,0.922921,0.865139)">

<search_score_summary>

<parameter name="nrs" value="-15"/>

<parameter name="nsi" value="0.9975"/>

<parameter name="nsm" value="2.9825"/>

<parameter name="nsp" value="20"/>

</search_score_summary>

</interprophet_result>

</analysis_result>

</search_hit>

</search_result>

</spectrum_query>

[David Shteynberg]

Hi Alex,

PTMProphet by default will ignore any spectrum with less than 90% probability (iProphet is used when available.)  You can change this by setting the option MINPROB= when you run the tool.  Let me know if that still fails for you.

Thanks,

-David

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[Alex Zelter]

Hi David,

Using MINPROB=0 solved this problem. Thanks!

Alex

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[David Shteynberg]

Great!  Although when you set MINPROB=0 the software is spending a lot of time evaluating PSMs you will never use, and some of them might have many mods causing a combinatorial expansion of peptide candidates the must be evaluated (although this is less of an issue for MASSDIFFMODE.) If you set this threshold a bit higher you can save yourself some time. Cheers!

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