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!
On Tue, Apr 7, 2020 at 1:13 PM Alex Zelter <[email protected]> wrote: > Hi David, > Using MINPROB=0 solved this problem. Thanks! > Alex > > On Monday, April 6, 2020 at 4:11:47 PM UTC-7, David Shteynberg wrote: >> >> 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 >> >> >> >> On Mon, Apr 6, 2020 at 3:27 PM Alex Zelter <[email protected]> wrote: >> >>> 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> >>> >>> -- >>> You received this message because you are subscribed to the Google >>> Groups "spctools-discuss" group. >>> To unsubscribe from this group and stop receiving 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