Hi Jesse, It appears to me that your msgfplus heavy search is not quiet doing what you want. The total mass of the variable mods listed near the top of the pepXML files should *include the mass of any static mod *on the same amino acid. It appears the msgfplus pepXML file is not doing this properly. Here is an snippet of the masses listed:
*<aminoacid_modification aminoacid="C" massdiff="125.047676" mass="228.0568604778" variable="N" description="Nethylmaleimide"/>* * <aminoacid_modification aminoacid="K" massdiff="8.014199" mass="136.1091620177" variable="N"/>* * <aminoacid_modification aminoacid="M" massdiff="15.994915" mass="147.0353996062" variable="Y" description="Oxidation"/>* * <aminoacid_modification aminoacid="Q" massdiff="-17.026548" mass="111.0320295114" peptide_terminus="n" variable="Y"/>* * <aminoacid_modification aminoacid="A" massdiff="42.010563" mass="113.0476767878" variable="Y"/>* * <aminoacid_modification aminoacid="S" massdiff="42.010563" mass="129.0425914099" variable="Y"/>* * <aminoacid_modification aminoacid="T" massdiff="42.010563" mass="143.0582414741" variable="Y"/>* * <aminoacid_modification aminoacid="V" massdiff="42.010563" mass="141.0789769162" variable="Y"/>* * <aminoacid_modification aminoacid="G" massdiff="42.010563" mass="99.0320267236" variable="Y"/>* * <aminoacid_modification aminoacid="N" massdiff="42.010563" mass="156.0534904472" variable="Y"/>* * <aminoacid_modification aminoacid="Q" massdiff="42.010563" mass="170.0691405114" variable="Y"/>* * <aminoacid_modification aminoacid="P" massdiff="42.010563" mass="139.063326852" variable="Y"/>* * <aminoacid_modification aminoacid="L" massdiff="42.010563" mass="155.0946269804" variable="Y"/>* * <aminoacid_modification aminoacid="I" massdiff="42.010563" mass="155.0946269804" variable="Y"/>* * <aminoacid_modification aminoacid="Y" massdiff="42.010563" mass="205.0738915383" variable="Y"/>* * <aminoacid_modification aminoacid="M" massdiff="42.010563" mass="173.0510476062" variable="Y"/>* * <aminoacid_modification aminoacid="W" massdiff="42.010563" mass="228.0898759535" variable="Y"/>* * <aminoacid_modification aminoacid="F" massdiff="42.010563" mass="189.0789769162" variable="Y"/>* * <aminoacid_modification aminoacid="C" massdiff="42.010563" mass="145.0197474778" variable="Y"/>* * <aminoacid_modification aminoacid="D" massdiff="42.010563" mass="157.037506032" variable="Y"/>* * <aminoacid_modification aminoacid="E" massdiff="42.010563" mass="171.0531560962" variable="Y"/>* * <aminoacid_modification aminoacid="H" massdiff="42.010563" mass="179.0694748624" variable="Y"/>* * <aminoacid_modification aminoacid="R" massdiff="42.010563" mass="198.1116740281" variable="Y"/>* * <aminoacid_modification aminoacid="K" massdiff="42.010563" mass="170.1055260177" variable="Y"/>* * <aminoacid_modification aminoacid="K" massdiff="114.04293" mass="242.1378930177" variable="Y" description="GlyGly"/>* This is the bug in the MSGF+ pepXML file which results in entries like this: *<modification_info modified_peptide="SKGIKSEPHSPGIPEIFRTA">* *<mod_aminoacid_mass position="2" mass="136.1091620177"/>* *<mod_aminoacid_mass position="5" mass="136.1091620177"/>* *<mod_aminoacid_mass position="5" mass="242.1378904877"/>* *</modification_info>* This entry is nonsensical in the world of pepXML as each position within a peptide should have only one mass. Your options are to alert the msgfplus developers to correct this bug. Or run the heavy search using a variable K mass of 114.04293 + 8.014199 . Cheers, -David P.S. Your use of +42.010563 (acetylation) on nearly every amino acid is probably overkill as it may unnecessarily slow your search. How many do you actually observe in your data? I didn't find any in you light search. Also, you may see higher sensitivity if you include decoys in you search database. On Tue, Dec 16, 2014 at 11:26 AM, Jesse <[email protected]> wrote: > > Yes I am using 4.8. I forgot to set variable metOx for the heavy and > PTM prophet now finishes, but none of the modifications are localized. I > used the following to account for heavy K and diglycine (actual file > structure removed): > > > > > > > > > > *cd c:/FILESTRUCTURE& c:\Inetpub\tpp-bin\PTMProphetParser > K,122.0572,M,15.995 MZTOL=0.1 NOUPDATE > c:/FILESTRUCTURE/UV0h.interact.ipro.pep.xml > UV0h.test122m16.interact.ptm.pep.xml I also tried running it with > K,114.043,M,15.995 and neither result has localized diglycine > modifications. Do I need to specify fixed modification of lysine to > account for silac? Thanks, Jesse * > On 12/16/2014 11:00 AM, David Shteynberg wrote: > > Are you using version 4.8 of the TPP? Can you send the commands (amino > acids and modifications) you are using for both the light that works and > heavy that doesn't? > > Thanks, > -David > > On Tue, Dec 16, 2014 at 10:29 AM, Jesse <[email protected]> wrote: >> >> David, >> >> Yes I have multiple pepXML files from heavy and light. I tried your >> suggestion of analyzing them separately, and PTMprophet works on the light >> pepXML, but with the heavy pepXML, PTMprophet does not finish and it >> returns many lines that look like this: >> >> WARNING: Illegal peptide with unknown mod: KKQLQDEMLRRV >> WARNING: Unrecognized mod on peptide: GGAPLSPQTHRFM[147]WARNING: Cannot >> initialize for sequence: GGAPLSPQTHRFM[147], unknown mods may exist in >> spectrum t02095_A.19205.19205.3WARNING: Illegal peptide with unknown mod: >> HDQFKSTLPDADREREA >> >> Warnings similar to the first and last line are reported during PTMprophet >> of the light pepXML, but warnings like the middle two only occurred with the >> heavy pepXML. >> >> Thanks for your help, >> Jesse >> >> >> >> On 12/15/2014 2:47 PM, David Shteynberg wrote: >> >> Hi Jesse, >> >> I am not certain I fully understand the issue, but it sounds to me like >> you have multiple searches and should therefore have multiple pepXML files >> (from you heavy and light). I would suggest you modify your pipeline >> slightly by analyzing the heavy and the light searches separately and >> combining them only after PTMProphet has processed the results. Is this >> feasible? >> >> Thanks, >> -David >> >> On Mon, Dec 15, 2014 at 2:28 PM, Jesse <[email protected]> wrote: >>> >>> David, >>> >>> I think I need to have some modifications added to the TPP code for >>> PTMprophet to work. I trying to combine two database searches that specify >>> either fixed light lysine or fixed heavy lysine (+8.0142), both with >>> variable modification of diGlycine (+114.0429). Therefore, lysine in my >>> datasets is found modified with +8.0142, +114.0429, or +122.0572. If you >>> add these to TPP, will I be able to combine these searches and validate the >>> mods with PTMprophet? >>> >>> Any help would be appreciated. >>> >>> Thank you, >>> Jesse >>> >>> On 8/12/2014 1:16 PM, David Shteynberg wrote: >>> >>>> You need to only enter the variable mods for PTMProphet. However, >>>> mods that are unknown will not be recognized without a change to the >>>> code. A future version of PTMProphet will hopefully correct these >>>> issues. >>>> >>>> -David >>>> >>>> On Tue, Aug 12, 2014 at 11:59 AM, Jesse Meyer <[email protected]> >>>> wrote: >>>> >>>>> I am also getting this error with a peptide containing a fixed >>>>> modification >>>>> of cysteine (N-ethylmaleimide +125.047679). I am running PTMprophet to >>>>> localize a different variable modification on lysine. Do I need to >>>>> enter >>>>> all mods including fixed mods under PTMprophet in the Petunia GUI? >>>>> >>>>> Thanks, >>>>> Jesse >>>>> >>>>> >>>>> On Saturday, July 12, 2014 7:13:57 AM UTC-7, [email protected] wrote: >>>>> >>>>>> Sure. Let me know what you need and I will send you the data. >>>>>> Best, >>>>>> Oded >>>>>> >>>>>> On Saturday, July 12, 2014 5:11:39 AM UTC+10, David Shteynberg wrote: >>>>>> >>>>>>> Hi Oded, >>>>>>> >>>>>>> >>>>>>> It is likely there is an issue with PTMProphet that doesn't recognize >>>>>>> the modifications in your dataset. If you can share a portion of >>>>>>> your >>>>>>> data for testing purposes I can help troubleshoot further. >>>>>>> >>>>>>> Thank you! >>>>>>> -David >>>>>>> >>>>>>> On Tue, Jul 1, 2014 at 5:33 AM, Oded <[email protected]> wrote: >>>>>>> >>>>>>>> Hi all, >>>>>>>> I am using TPP 4.7.1 on Win7 and currently trying to analyze data of >>>>>>>> dimethylated phosphopeptides searched with X!tandem. >>>>>>>> The searches were done by using 2 sets of fixed modifications for >>>>>>>> the >>>>>>>> Cys >>>>>>>> alkylation plus light or heavy dimethylation on N-term and K with >>>>>>>> STY >>>>>>>> phosphorylation, Met-Ox and protein N-term acetylation as set as >>>>>>>> variable >>>>>>>> modifications. >>>>>>>> When I try to run PTM prophet (following peptide prophet and >>>>>>>> iprophet >>>>>>>> runs) >>>>>>>> I getting the following error: >>>>>>>> >>>>>>>> >>>>>>>> EXECUTING: run_in >>>>>>>> c:/Inetpub/wwwroot/ISB/data/Oded/Enzo/Phospho_June2014; >>>>>>>> c:\Inetpub\tpp-bin\PTMProphetParser >>>>>>>> STY,79.966,C,57.021464,n,34.063117,K,34.063117,M,15.99 MZTOL=0.1 >>>>>>>> >>>>>>>> >>>>>>>> c:/Inetpub/wwwroot/ISB/data/Oded/Enzo/Phospho_June2014/interactNative1.ipro.pep.xml >>>>>>>> interactNative >>>>>>>> INFO: Writing file interactNative1.ptm.pep.xml ... >>>>>>>> INFO: Reading file >>>>>>>> >>>>>>>> >>>>>>>> c:/Inetpub/wwwroot/ISB/data/Oded/Enzo/Phospho_June2014/interactNative1.ipro.pep.xml >>>>>>>> ... >>>>>>>> WARNING: Cannot initialize for sequence: n[71]M[147]KDEPRSTNLFMK, >>>>>>>> unknown >>>>>>>> mods may exist in spectrum CHH20140618_M1HTiO2_100mM.03738.03738.2 >>>>>>>> WARNING: Cannot initialize for sequence: n[35]K[162]S[167]PAAAR, >>>>>>>> unknown >>>>>>>> mods may exist in spectrum CHH20140618_M1HTiO2_100mM.03900.03900.2 >>>>>>>> WARNING: Cannot initialize for sequence: n[35]VPS[167]RHINIGR, >>>>>>>> unknown >>>>>>>> mods >>>>>>>> may exist in spectrum CHH20140618_M1HTiO2_100mM.04358.04358.2 >>>>>>>> WARNING: Cannot initialize for sequence: >>>>>>>> n[35]EGEEPT[181]VYSDEEEPK[162]DESAR, unknown mods may exist in >>>>>>>> spectrum >>>>>>>> CHH20140618_M1HTiO2_100mM.04499.04499.3 >>>>>>>> WARNING: Cannot initialize for sequence: n[35]GTPGPAVR, unknown mods >>>>>>>> may >>>>>>>> exist in spectrum CHH20140618_M1HTiO2_100mM.05584.05584.2 >>>>>>>> WARNING: Unrecognized mod on peptide: >>>>>>>> n[35]SCFESS[167]PDPELK[162] >>>>>>>> WARNING: Unrecognized mod on peptide: >>>>>>>> n[35]SCFESS[167]PDPELK[162] >>>>>>>> WARNING: Unrecognized mod on peptide: >>>>>>>> n[35]SCFESS[167]PDPELK[162] >>>>>>>> WARNING: Unrecognized mod on peptide: >>>>>>>> n[35]SCFESS[167]PDPELK[162] >>>>>>>> WARNING: Unrecognized mod on peptide: >>>>>>>> n[35]SCFESS[167]PDPELK[162] >>>>>>>> WARNING: Unrecognized mod on peptide: >>>>>>>> n[35]SCFESS[167]PDPELK[162] >>>>>>>> {this appear 100-200 times} >>>>>>>> >>>>>>>> >>>>>>>> >>>>>>>> This application has requested the Runtime to terminate it in an >>>>>>>> unusual >>>>>>>> way. >>>>>>>> Please contact the application's support team for more information. >>>>>>>> >>>>>>>> command "c:\Inetpub\tpp-bin\PTMProphetParser >>>>>>>> STY,79.966,C,57.021464,n,34.063117,K,34.063117,M,15.99 MZTOL=0.1 >>>>>>>> >>>>>>>> >>>>>>>> c:/Inetpub/wwwroot/ISB/data/Oded/Enzo/Phospho_June2014/interactNative1.ipro.pep.xml >>>>>>>> interactNative1.ptm.pep.xml" failed: No such process >>>>>>>> >>>>>>>> >>>>>>>> I tried adding both heavy and light dimethylation (+28 and +34 on K >>>>>>>> and >>>>>>>> N-term) just light and just heavy (as shown above), the the mass of >>>>>>>> the >>>>>>>> DiMet N-term as it appear in the pepXML (+35 or +29) but the error >>>>>>>> is >>>>>>>> still >>>>>>>> the same... >>>>>>>> Is there a way to sort this? >>>>>>>> Many thanks, >>>>>>>> Oded >>>>>>>> >>>>>>>> -- >>>>>>>> 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 post to this group, send email to [email protected]. >>>>>>>> Visit this group at http://groups.google.com/group/spctools-discuss >>>>>>>> . >>>>>>>> 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. >>>>> To unsubscribe from this group and stop receiving emails from it, send >>>>> an >>>>> email to [email protected]. >>>>> To post to this group, send email to [email protected] >>>>> . >>>>> Visit this group at http://groups.google.com/group/spctools-discuss. >>>>> For more options, visit https://groups.google.com/d/optout. >>>>> >>>> >>> >>> -- >>> Jesse Meyer >>> Graduate Student >>> Department of Chemistry and Biochemistry >>> University of California, San Diego >>> La Jolla, CA 92092 >>> >>> >>> --- >>> This email is free from viruses and malware because avast! Antivirus >>> protection is active. >>> http://www.avast.com >>> >>> -- >>> 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 post to this group, send email to [email protected]. >>> Visit this group at http://groups.google.com/group/spctools-discuss. >>> 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. >> To unsubscribe from this group and stop receiving emails from it, send an >> email to [email protected]. >> To post to this group, send email to [email protected]. >> Visit this group at http://groups.google.com/group/spctools-discuss. >> For more options, visit https://groups.google.com/d/optout. >> >> >> >> -- >> Jesse Meyer >> Graduate Student >> Department of Chemistry and Biochemistry >> University of California, San Diego >> La Jolla, CA 92092 >> >> >> >> ------------------------------ >> <http://www.avast.com/> >> >> This email is free from viruses and malware because avast! Antivirus >> <http://www.avast.com/> protection is active. >> >> -- >> 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 post to this group, send email to [email protected]. >> Visit this group at http://groups.google.com/group/spctools-discuss. >> 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. > To unsubscribe from this group and stop receiving emails from it, send an > email to [email protected]. > To post to this group, send email to [email protected]. > Visit this group at http://groups.google.com/group/spctools-discuss. > For more options, visit https://groups.google.com/d/optout. > > > > -- > Jesse Meyer > Graduate Student > Department of Chemistry and Biochemistry > University of California, San Diego > La Jolla, CA 92092 > > > > ------------------------------ > <http://www.avast.com/> > > This email is free from viruses and malware because avast! Antivirus > <http://www.avast.com/> protection is active. > > -- > 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 post to this group, send email to [email protected]. > Visit this group at http://groups.google.com/group/spctools-discuss. > 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. To unsubscribe from this group and stop receiving emails from it, send an email to [email protected]. To post to this group, send email to [email protected]. Visit this group at http://groups.google.com/group/spctools-discuss. For more options, visit https://groups.google.com/d/optout.
