Thanks Jimmy for your prompt response. I do see that for isoform P06396-2, the preceding methionine is the first residue of the sequence and I used both comet & tandem for the database search.
For Comet search, I did have the "clip_nterm_methionine <http://comet-ms.sourceforge.net/parameters/parameters_201801/clip_nterm_methionine.php>" parameter set to "1" so you explanation makes sense. But for Tandem search, I am not sure if Tandem has an equivalent parameter. But i can see that the tandem peptideprophet pepxml also identifies this peptide as unique to isoform P06396-2. <spectrum_query spectrum="XL_150506_IDA_PDAY_F7_proteinpilot.09460.09460.2" start_scan="9460" end_scan="9460" precursor_neutral_mass="1096.5930" assumed_charge="2" index="28557" retention_time_sec="2248.41"> <search_result> <search_hit hit_rank="1" peptide="VVEHPEFLK" peptide_prev_aa="M" peptide_next_aa="A" protein="sp|P06396-2|GELS_HUMAN" protein_descr="Isoform 2 of Gelsolin OS=Homo sapiens OX=9606 GN=GSN" num_tot_proteins="1" num_matched_ions="17" tot_num_ions="16" calc_neutral_pep_mass="1096.5917" massdiff="0.001" num_tol_term="2" num_missed_cleavages="0" is_rejected="0"> Do you know if there is a parameter similar to 'clip_nterm_methionine' in tandem as well? and Does RefreshParser have any parameters to control this behavior? Many Thanks Vidya On Wed, Jun 5, 2019 at 11:27 AM Jimmy Eng <jke...@gmail.com> wrote: > Without more info, here's my educated guess at an explanation: that > peptide is not a fully tryptic peptide in the proteins you list as it is > preceded by a methionine residue. For isoform P06396-2, the preceding > methionine is the first residue of the sequence and I'm assuming you did a > Comet search with the "clip_nterm_methionine > <http://comet-ms.sourceforge.net/parameters/parameters_201801/clip_nterm_methionine.php>" > parameter set to "1". With that start methionine removed, this allowed > that peptide to be fully tryptic in that particular isoform hence only that > isoform is listed as a matched protein. The enzyme context does matter > with RefreshParser; it's simply not a peptide string match. > > On Wed, Jun 5, 2019 at 11:15 AM Vidya Venkatraman <vidya.venk...@gmail.com> > wrote: > >> Hi Everyone, >> >> I am trying to run xinteract with the -D option so that RefreshParser can >> use the fasta database to find all proteins corresponding to identified >> peptides. >> >> This is the command I am using: >> xinteract -OARPwd -dDECOY_ -PPM -nR >> -D/scratch/venkatramanv/iphronesis/reference_files/Databases/UP_Human_Rev_Can+Iso_20190410_DECOY.fasta >> -N/scratch/venkatramanv/iphronesis/platform_workspace/jobs/SJOB2122/peptideprophet/2122_PDAY_DDA_Isoforms.comet.interact.pep.xml >> *.comet.pep.xml >> >> Despite using the -D database option, i noticed that some peptides in the >> interact.pep.xml are matched uniquely to a specific isoform (even though >> that peptide is not proteotypic or unique to any isoform). >> >> For example: >> >> <spectrum_query >> spectrum="XL_150506_IDA_PDAY_F8_proteinpilot.08799.08799.2" >> start_scan="8799" end_scan="8799" precursor_neutral_mass="1096.592165" >> assumed_charge="2" index="20868" retention_time_sec="2254.3"> >> <search_result> >> <search_hit hit_rank="1" peptide="VVEHPEFLK" peptide_prev_aa="M" >> peptide_next_aa="A" protein="sp|P06396-2|GELS_HUMAN" num_tot_proteins="1" >> num_matched_ions="11" tot_num_ions="16" calc_neutral_pep_mass="1096.591696" >> massdiff="0.000470" num_tol_term="1" num_missed_cleavages="0" >> num_matched_peptides="6184"> >> <search_score name="xcorr" value="2.205"/> >> <search_score name="deltacn" value="0.575"/> >> <search_score name="deltacnstar" value="0.000"/> >> <search_score name="spscore" value="350.4"/> >> <search_score name="sprank" value="1"/> >> <search_score name="expect" value="4.94E-03"/> >> <analysis_result analysis="peptideprophet"> >> <peptideprophet_result probability="1.0000" >> all_ntt_prob="(0.0000,1.0000,1.0000)"> >> <search_score_summary> >> <parameter name="fval" value="5.5754"/> >> <parameter name="ntt" value="1"/> >> <parameter name="nmc" value="0"/> >> <parameter name="massd" value="0.429"/> >> <parameter name="isomassd" value="0"/> >> <parameter name="RT" value="1372.21"/> >> <parameter name="RT_score" value="0.02"/> >> </search_score_summary> >> </peptideprophet_result> >> </analysis_result> >> </search_hit> >> </search_result> >> </spectrum_query> >> >> See attached the sequence alignment of all isoforms of GELS_HUMAN, you >> can see that this peptide matches the canonical form as well as all >> isoforms hence the num_tot_proteins should be = "4" and protein should >> be ="sp|P06396|GELS_HUMAN" with <alternative_protein protein=" >> sp|P06396-2|GELS_HUMAN"/>, >> <alternative_protein protein="sp|P06396-3|GELS_HUMAN"/> , >> <alternative_protein protein="sp|P06396-4|GELS_HUMAN"/> >> >> I am happy to share all the comet.pep.xml & interact.pep.xml files along >> with the fasta database via Box folder. >> >> Can someone please help me understand if this is expected behavior? if >> so, how i can fix this? >> >> Regards >> Vidya >> >> -- >> 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-discuss+unsubscr...@googlegroups.com. >> To post to this group, send email to spctools-discuss@googlegroups.com. >> Visit this group at https://groups.google.com/group/spctools-discuss. >> To view this discussion on the web visit >> https://groups.google.com/d/msgid/spctools-discuss/6b292366-55e1-4fe2-9994-ea9fff3d97a9%40googlegroups.com >> <https://groups.google.com/d/msgid/spctools-discuss/6b292366-55e1-4fe2-9994-ea9fff3d97a9%40googlegroups.com?utm_medium=email&utm_source=footer> >> . >> For more options, visit https://groups.google.com/d/optout. >> > -- > You received this message because you are subscribed to a topic in the > Google Groups "spctools-discuss" group. > To unsubscribe from this topic, visit > https://groups.google.com/d/topic/spctools-discuss/Iq4P_h0DizM/unsubscribe > . > To unsubscribe from this group and all its topics, send an email to > spctools-discuss+unsubscr...@googlegroups.com. > To post to this group, send email to spctools-discuss@googlegroups.com. > Visit this group at https://groups.google.com/group/spctools-discuss. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/CAJqD6ENwEimtBu3twOwHUPzSQ_m0Q2bzNpqiYcghbpaoRqPUkA%40mail.gmail.com > <https://groups.google.com/d/msgid/spctools-discuss/CAJqD6ENwEimtBu3twOwHUPzSQ_m0Q2bzNpqiYcghbpaoRqPUkA%40mail.gmail.com?utm_medium=email&utm_source=footer> > . > For more options, visit https://groups.google.com/d/optout. > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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