Hi Brian, It is possible something is broken in 4.7. However, first can you double check the command foir your n-terminal mass which should be n49.0924 since that would match the mass of the heavy label in the search? If that still doesn't work please send the file to me so I can debug it. BTW, if you are using monoisotopic masses during the search specifying them on the commandline is not required.
Best regards, -David On Sun, Jul 6, 2014 at 3:26 PM, Brian Hampton <[email protected]> wrote: > Hello, > > I am having difficulty getting ASAPRatio in TPP 4.7.0 to use the corrrect > m/z when analyzing chemical isotopic labeled peptides (D0 or D6 Acetone via > reductive alkylation). > > Instead of choosing the +6 partner of the light label it instead chooses +48 > which is the mass of the heavy label. In the interact.pep.xml file it has > correctly identified the labels and their corresponding mass differences, > but for some reason isn't using the massdiff but rather the label mass for > the heavy label (which was the variable mod in the search). I've searched > this as before when it worked e.g. I set the light label to be a static > modification. I then set the difference between the heavy and light > modification as a variable mod. The Tandem (or Comet) search results > correctly identify pairs of peptides with D0 and D6 modified n-term and K > residues. In the ASAPRatio part of the form in Petunia, I select n and K as > the modified residues, and then I specify the monoisotopic masses for each > of the modified residues that are present in the sample e.g. n, K, M, & C. > The ASAPRatio results show a mass difference of (for +1 charge state) +96, > (for +2 charge state) +48 where the CID was performed on the light peptide > and conversely mass difference of (for +1 charge state) +48 (for +2 charge > state) +24 where the CID was performed on the heavy peptide. > > This had been working correctly in TPP 4.6.1 and .2, but not anymore in > 4.7.0. Though 4.7.0 doesn't seem to be working with chemical isotopic > labeling, it does work with SILAC data. > > > Here is a snip from the interact.pep.xml file showing the modifications it > is ~using: > > <search_summary > base_name="/usr/local/tpp/data/projects/s-mouse-plasma/medres/140627-s-mouse-serum-7986" > search_engine="X! Tandem (k-score)" precursor_mass_type="monoisotopic" > fragment_mass_type="monoisotopic" search_id="1"> > <search_database > local_path="/usr/local/tpp/data/dbase/mouse-1404-crap_DECOY.fasta" > type="AA"/> > <enzymatic_search_constraint enzyme="trypsin" > max_num_internal_cleavages="2" min_number_termini="2" /> > <aminoacid_modification aminoacid="C" massdiff="57.0215" > mass="160.0306" variable="N" /> > <aminoacid_modification aminoacid="K" massdiff="42.0470" > mass="170.1419" variable="N" /> > <aminoacid_modification aminoacid="C" massdiff="-17.0265" > mass="143.0041" variable="Y" symbol="^" /><!--X! Tandem n-terminal AA > variable modification--> > <aminoacid_modification aminoacid="E" massdiff="-18.0106" > mass="111.0320" variable="Y" symbol="^" /><!--X! Tandem n-terminal AA > variable modification--> > <aminoacid_modification aminoacid="K" massdiff="6.0377" > mass="176.1796" variable="Y" /> > <aminoacid_modification aminoacid="M" massdiff="15.9949" > mass="147.0354" variable="Y" /> > <aminoacid_modification aminoacid="Q" massdiff="-17.0265" > mass="111.0321" variable="Y" symbol="^" /><!--X! Tandem n-terminal AA > variable modification--> > <terminal_modification terminus="n" massdiff="42.0470" > mass="43.0548" protein_terminus="N" variable="N" /> > <terminal_modification terminus="n" massdiff="42.0106" > mass="85.0654" protein_terminus="N" variable="Y" symbol="^" /><!--X! Tandem > n-terminal AA variable modification--> > <terminal_modification terminus="n" massdiff="6.0377" > mass="49.0924" protein_terminus="N" variable="Y" /> > > > Command to start Xinteract: > > EXECUTING: cd /usr/local/tpp/data/projects/s-mouse-plasma/medres; > /usr/local/tpp/bin/xinteract -Ninteract-tandem-statlight-varheavy.pep.xml > -p0.0 -l7 -Op -dDECOY > -A-lnK-b-F-C-r0.2-mn48.08461K176.179566M147.035395C160.030654 > 140627-s-mouse-serum-7986.tandem.pep.xml > > Any help greatly appreciated. > > Thanks in advance, > Brian > > -- > 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.
