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.
