Hi. I'm running into an issue with the TPP/Kojak pipeline. I am running a crosslinking analysis using Kojak. As you know, Kojak also reports unlinked PSMs. I'm finding that PeptideProphet is filtering out ALL the unlinked PSMs in my analysis. If I process the dataset with Kojak/Percolator I get *8509 unlinked PSMs* and 585 crosslinked PSMs with a q value <= 0.05 Using Kojak/TPP v5.2.1-dev Flammagenitus, Build 202001280920-8004 I get *0 unlinked PSMs* and 564 crosslinked PSMs with a PeptideProphet error rate of >= 0.95 Using Kojak/TPP v5.2.0 Flammagenitus, Build 202001311412-exported I also get *0 unlinked PSMs* and 564 crosslinked PSMs with a PeptideProphet error rate of >= 0.95
If I look at the raw data for a specific unlinked PSM, I find things like this in the Kojak pep.xml file spectrum_query spectrum="UWPRLumos_2020_0124_AZ_012_AZ866_xlink05.24437.24437.3" start_scan="24437" end_scan="24437" precursor_neutral_mass="1782.949616" assumed_charge="3" index="25666" retention_time_sec="91.8"> <search_result> <search_hit hit_rank="1" peptide="KPIDYTILDDIGHGVK" peptide_prev_aa="R" peptide_next_aa="V" protein="hAbi2_1-158" protein_link_pos_a="139" num_tot_proteins="1" calc_neutral_pep_mass="1782.951515" massdiff="0.001899" xlink_type="na" num_tol_term="2" num_missed_cleavages="0"> <search_score name="kojak_score" value="5.9750"/> <search_score name="delta_score" value="5.0900"/> <search_score name="ppm_error" value="1.0650"/> <search_score name="e-value" value="3.950e-13"/> <search_score name="ion_match" value="42"/> <search_score name="consecutive_ion_match" value="13"/> <analysis_result analysis="peptideprophet"> <peptideprophet_result probability="0" all_ntt_prob="(0,0,0)" analysis="incomplete"> <search_score_summary> <parameter name="fval" value="7.7950"/> <parameter name="ntt" value="2"/> <parameter name="nmc" value="0"/> <parameter name="massd" value="1.065"/> <parameter name="isomassd" value="0"/> </search_score_summary> </peptideprophet_result> so scan 24437 identifying KPIDYTILDDIGHGVK gets a e value of 3.950e-13 from kojak, which is good, but gets a probability of 0 from peptideprophet (see below). A comet/percolator analysis shows a comet e value of 1.67E-14 and a percolator q-value of 0.00009243 for the same PSM (both very good) A kojak/percolator analysis shows a Kojak e value of 3.950e-13 and a percolator q value of 0.000001555 for the same PSM. Again, both very good. Looking at interact.pep.xml I see: <spectrum_query spectrum="UWPRLumos_2020_0124_AZ_012_AZ866_xlink05.24437.24437.3" start_scan="24437" end_scan="24437" precursor_neutral_mass="1782.949616" assumed_charge="3" index="25666" retention_time_sec="91.8"> <search_result> <search_hit hit_rank="1" peptide="KPIDYTILDDIGHGVK" peptide_prev_aa="R" peptide_next_aa="V" protein="hAbi2_1-158" protein_link_pos_a="139" num_tot_proteins="1" calc_neutral_pep_mass="1782.951515" massdiff="0.001899" xlink_type="na" num_tol_term="2" num_missed_cleavages="0"> <search_score name="kojak_score" value="5.9750"/> <search_score name="delta_score" value="5.0900"/> <search_score name="ppm_error" value="1.0650"/> <search_score name="e-value" value="3.950e-13"/> <search_score name="ion_match" value="42"/> <search_score name="consecutive_ion_match" value="13"/> <analysis_result analysis="peptideprophet"> <peptideprophet_result probability="0" all_ntt_prob="(0,0,0)" analysis="incomplete"> <search_score_summary> <parameter name="fval" value="7.7950"/> <parameter name="ntt" value="2"/> <parameter name="nmc" value="0"/> <parameter name="massd" value="1.065"/> <parameter name="isomassd" value="0"/> </search_score_summary> </peptideprophet_result> </analysis_result> </search_hit> </search_result> </spectrum_query> So I am confused why PeptideProphet is assigning all unlinked peptides in these data with a probability of 0. I tried the latest stable version of TPP as well as the latest daily build. I see the same results in each. Any ideas about this would be very much appreciated. Thanks very much in advance! Alex -- 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 [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/52dce033-f9f8-4abf-aeeb-9f30ab5cdaf3%40googlegroups.com.
