Hi all, I have been trying to quantify our dimethyl labeled samples for the last week using TPP. I got it to work when I ran heavy and light label SEQUEST searches separate and in ASAP ratio I chose the option of "static modification quantification" and chose K and n-terminus as the "labeled residues" . I chose the option of " use fixed scan range for light and heavy". Then set m/z range to "0.05". At this point I put monoisotopic mass of K with the dimethyl heavy label (164.01706) to the "specified residue mass". I tried adding n- terminus as 36.0757 but it says I have three different masses for n- terminus (29.0388, 36.0757 and 37.0838) so I used only K with dimethylation (heavy). It runs without a hitch but during the run it says n terminus is labeled with 29.0388 and 37.0838. Why does is give a protonated N-terminus for heavy and light label ? K seems to have the right numbers for heavy and light labels.
I am using windows 7, TPP v 4.3 jetstream rev 1 build 200909091257 (MinGW) and we are using an LTQ Orbitrap. The command line is below, I would really appreciate all the help. cheers Gogce Crynen # Commands for session 9UNL73SYC on Tue Jan 11 13:12:34 2011 # BEGIN COMMAND BLOCK ###### BEGIN Command Execution ###### [Tue Jan 11 13:12:34 2011] EXECUTING: run_in c:/Inetpub/wwwroot/ISB/ data/111610_crudeLYSATE_SIDL_1to1/HandL; c:\Inetpub\tpp-bin\xinteract -N111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml -p0.05 -l7 -OAp -A- lKn-F-r0.05-S-mK164.1706 c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_H.pep.xml c:/Inetpub/wwwroot/ISB/ data/111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L.pep.xml OUTPUT: c:\Inetpub\tpp-bin\xinteract (TPP v4.3 JETSTREAM rev 1, Build 200909091257 (MinGW)) running: "C:/Inetpub/tpp-bin/InteractParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" "c:/Inetpub/wwwroot/ ISB/data/111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_H.pep.xml" "c:/Inetpub/wwwroot/ISB/ data/111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L.pep.xml" -L"7"" file 1: c:/Inetpub/wwwroot/ISB/data/111610_crudeLYSATE_SIDL_1to1/ HandL/111610_crudeLYSATE_SIDL_1to1_Rep1_H.pep.xml file 2: c:/Inetpub/wwwroot/ISB/data/111610_crudeLYSATE_SIDL_1to1/ HandL/111610_crudeLYSATE_SIDL_1to1_Rep1_L.pep.xml processed altogether 34720 results results written to file c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml direct your browser to http://localhost/ISB/data/111610_crudeLYSATE_SIDL_1to1/HandL/111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.shtml command completed in 6 sec running: "C:/Inetpub/tpp-bin/PeptideProphetParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" MINPROB=0.05 ACCMASS" using Accurate Mass Bins (SEQUEST) adding Accurate Mass mixture distr init with SEQUEST trypsin MS Instrument info: Manufacturer: Thermo Scientific, Model: LTQ Orbitrap, Ionization: ESI, Analyzer: FTMS, Detector: unknown PeptideProphet (TPP v4.3 JETSTREAM rev 1, Build 200909091257 (MinGW)) akel...@isb read in 71 1+, 9098 2+, 7422 3+, 228 4+, 22 5+, 0 6+, and 0 7+ spectra. Initialising statistical models ... Iterations: .........10.........20.........30... WARNING: Mixture model quality test failed for charge (1+). model complete after 34 iterations command completed in 21 sec running: "C:/Inetpub/tpp-bin/ProphetModels.pl -i 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" Analyzing 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml ... Reading Accurate Mass Model model +1 ... Reading Accurate Mass Model model +2 ... Reading Accurate Mass Model model +3 ... Reading Accurate Mass Model model +4 ... Reading Accurate Mass Model model +5 ... Reading Accurate Mass Model model +6 ... Reading Accurate Mass Model model +7 ... Parsing search results "c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/111610_crudeLYSATE_SIDL_1to1_Rep1_H (SEQUEST)"... => Total of 999 hits. Parsing search results "c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/111610_crudeLYSATE_SIDL_1to1_Rep1_L (SEQUEST)"... => Total of 836 hits. command completed in 1 sec running: "C:/Inetpub/tpp-bin/ASAPRatioPeptideParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" -lKn -F -r0.05 -S - mK164.1706" K 156.126 164.171 n 29.0388 37.0838 static heavy label for file c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_H.mzXML static light label for file c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L.mzXML command completed in 35 sec running: "C:/Inetpub/tpp-bin/DatabaseParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml"" command completed in 1 sec running: "C:/Inetpub/tpp-bin/RefreshParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" "C:\Xcalibur\database \ipi.MOUSE.v3.72.fasta"" - Searching the tree... - Linking duplicate entries... - Printing results... - Building Commentz-Walter keyword tree...command completed in 4 sec running: "c:/Inetpub/wwwroot/../tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/ wwwroot/ISB/data/111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" command completed in 1 sec running: "C:/Inetpub/tpp-bin/ProteinProphet "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml" XML" ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v4.3 JETSTREAM rev 1, Build 200909091257 (MinGW)) (xml input) (report Protein Length) (using degen pep info) . . . reading in c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml. . . . . . read in 14 1+, 1011 2+, 752 3+, 21 4+, 2 5+, 0 6+, 0 7+ spectra with min prob 0.05 INFO: mu=3.55413e-005, db_size=51125798 protein probabilities written to file c:/Inetpub/wwwroot/ISB/data/ 111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml direct your browser to http://localhost/ISB/data/111610_crudeLYSATE_SIDL_1to1/HandL/111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.shtml command completed in 3 sec running: "C:/Inetpub/tpp-bin/ProtProphModels.pl -i 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml" Analyzing 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml ... command completed in 0 sec running: "C:/Inetpub/tpp-bin/ASAPRatioProteinRatioParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml"" command completed in 111 sec running: "C:/Inetpub/tpp-bin/ASAPRatioPvalueParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml"" command completed in 2 sec C:/Inetpub/tpp-bin/InteractParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" "c:/Inetpub/wwwroot/ ISB/data/111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_H.pep.xml" "c:/Inetpub/wwwroot/ISB/ data/111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L.pep.xml" -L"7" 6 sec C:/Inetpub/tpp-bin/PeptideProphetParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" MINPROB=0.05 ACCMASS 21 sec C:/Inetpub/tpp-bin/ProphetModels.pl -i 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml 1 sec C:/Inetpub/tpp-bin/ASAPRatioPeptideParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" -lKn -F -r0.05 -S - mK164.1706 35 sec C:/Inetpub/tpp-bin/DatabaseParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" C:/Inetpub/tpp-bin/RefreshParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" "C:\Xcalibur\database \ipi.MOUSE.v3.72.fasta" 4 sec c:/Inetpub/wwwroot/../tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/wwwroot/ ISB/data/111610_crudeLYSATE_SIDL_1to1/HandL/ 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml 1 sec C:/Inetpub/tpp-bin/ProteinProphet "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.pep.xml" "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml" XML 3 sec C:/Inetpub/tpp-bin/ProtProphModels.pl -i 111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml 0 sec C:/Inetpub/tpp-bin/ASAPRatioProteinRatioParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml" 111 sec C:/Inetpub/tpp-bin/ASAPRatioPvalueParser "111610_crudeLYSATE_SIDL_1to1_Rep1_L_H2.prot.xml" 2 sec job completed in 185 sec END OUTPUT RETURN CODE:0 ###### End Command Execution ###### # All finished at Tue Jan 11 13:15:39 2011 # END COMMAND BLOCK -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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