Hi Alberto and Luis,
After reading your posts, I think that you could help me.
At this time, I'm using TPP for iTRAQ quantitation after a search with
X!Tandem as you did.
I'm facing some problems with the time of the search. In my case, I use
1 static modification and I'd like to include seven others potential
modifications.
With only one static and one or two potential, it's quite faste (around
20-30 min on my system 2 processors, 8 virtual cores). As soon as I
increase the number of potential modif the time increase dramatically.
Did you observe such issue ? If not, do you use a refinement search ?
I'm wondering if it's only a matter of power of my computer or if it's
rather due to X!Tandem algorithm.
Thanks in advance for any advice.
Fred
Le 9/22/2014 7:55 PM, Alberto Labarga a écrit :
Hi Luis,
thanks for the useful insight, I guess for iTRAQ(8) we need to use
<note>residue modification parameters</note>
<note type="input" label="residue, modification mass">46.9955457863@C,
304.205360@[,304.205360@K</note>
<note type="input" label="residue, potential modification
mass">15.994915@M,304.205360@Y</note>
is that correct? thanks a lot,
Alberto
El martes, 11 de octubre de 2011 20:30:27 UTC+2, Luis escribió:
Hello Marcus,
As luck would have it, I just had to conduct analysis of an
iTRAQ(8) sample using X!Tandem.
First, there is unfortunately no GUI to set the parameters for the
search, at least within the TPP.
Do make sure that you specify the appropriate modifications based
on the chemistry you used to prepare the peptides. Other than
alkylation and MMTS, you should add the isobaric mass tag to all
peptides -- typically at the N-term as well as Lysines, but this
might depend on the exact protocol you followed. Add the
following to the "residue, modification mass" tag:
144.102063@[,144.102063@K . A less abundant potential
modification on Y might be useful to add, under "residue,
potential modification mass", include 144.102063@Y , as well as
any other potential mods (e.g. Oxidized M, etc).
Also, note that the iTRAQ8-plex reagent has a very different mass
(304.205360), should you ever use that label.
The standard xtandem/k-score parameters that we ship with the TPP
already exclude fragment ions lower than 125.0 Daltons; you can
change this parameter by adding the following line to your params
file, just replace the value with your desired minimum m/z:
<note type="input" label="spectrum, minimum fragment mz">125.0</note>
Hope this helps with your searches,
--Luis
On Mon, Sep 26, 2011 at 11:33 PM, Marcus Sjödin
<[email protected] <javascript:>> wrote:
Hi!
I wish to run some iTRAQ labeled data through the TPP using
the embedded search engine xtandem! The peptides have been
alkylated using MMTS and labeled with the iTRAQ 4-plex
(114-117). The data was acquired on a Waters Synapt using DDA
mode. I have converted the .raw data to both mzML and mxXML
format and thus have the data in the format ready to use for
the database searching. My question regards how to define the
search tandem parameter file to be set up using isobaric
labeling?
Firstly, the default parameter file is set up for
carbamidomethylation but I need to change it to MMTS which I
assume just needs to be changed from
<note label="residue, modification mass"
type="input">57.021464@C</note>
into
<note label="residue, modification mass"
type="input">45.987721@C</note>
But how do I define the search for iTRAQ? I assume, a mass
addition for the isobaric label is needed for the MS scan as
well as a exclusion of 114-117 in the MS/MS scan is needed? Is
there any easy way of doing this? Is there a GUI to define the
correct search settings for this that works with the xtandem
in TPP? I would greatly appreciate any help on this topic!
Sincerely,
Marcus Sjödin
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