Hi Luis!
Thank you so much for the reply!
I have now changed the settings in tandem_params.xml to the following:
<note type="input" label="residue, modification mass">45.987721@C,
144.102063@[,144.102063@K</note>
<note type="input" label="residue, potential modification
mass">15.994915@M,144.102063@Y</note>
<note type="input" label="spectrum, minimum fragment mz">100.0</note>
Additionlay, I am not sure how to set the mass tolerance. I am using a
High resolution Synapt QTof that should handle at least 50 ppm as
precursor and 0.05 Da as fragments.
This is how param file look like:
<note> PRECURSOR MASS TOLERANCES. In the example below, a -2.0 Da to
4.0 Da (monoisotopic mass) window is searched for peptide candidates.
Since this is monoisotopic mass, so for non-accurate-mass instruments,
for which the precursor is often taken nearer to the isotopically
averaged mass, an asymmetric tolerance (-2.0 Da to 4.0 Da) is
preferable. This somewhat imitates a (-3.0 Da to 3.0 Da) window for
averaged mass (but not exactly)</note>
<note type="input" label="spectrum, parent monoisotopic mass error
minus">2.0</note>
<note type="input" label="spectrum, parent monoisotopic mass error
plus">4.0</note>
<note type="input" label="spectrum, parent monoisotopic mass error
units">Daltons</note>
<note>The value for this parameter may be 'Daltons' or 'ppm':
all
other values are ignored</note>
<note type="input" label="spectrum, parent monoisotopic mass isotope
error">no</note>
<note>This allows peptide candidates in windows around -1 Da
and -2
Da from the acquired mass to be considered. Only applicable when the
minus/plus window above is set to less than 0.5 Da. Good for accurate-
mass instruments for which the reported precursor mass is not
corrected to the monoisotopic mass. </note>
Should these changes fullfill my requirements or should I just leave
it? :
<note> PRECURSOR MASS TOLERANCES. In the example below, a -2.0 Da to
4.0 Da (monoisotopic mass) window is searched for peptide candidates.
Since this is monoisotopic mass, so for non-accurate-mass instruments,
for which the precursor is often taken nearer to the isotopically
averaged mass, an asymmetric tolerance (-2.0 Da to 4.0 Da) is
preferable. This somewhat imitates a (-3.0 Da to 3.0 Da) window for
averaged mass (but not exactly)</note>
<note type="input" label="spectrum, parent monoisotopic mass error
minus">25</note>
<note type="input" label="spectrum, parent monoisotopic mass error
plus">25</note>
<note type="input" label="spectrum, parent monoisotopic mass error
units">ppm</note>
<note>The value for this parameter may be 'Daltons' or 'ppm':
all
other values are ignored</note>
<note type="input" label="spectrum, parent monoisotopic mass isotope
error">yes</note>
<note>This allows peptide candidates in windows around -1 Da
and -2
Da from the acquired mass to be considered. Only applicable when the
minus/plus window above is set to less than 0.5 Da. Good for accurate-
mass instruments for which the reported precursor mass is not
corrected to the monoisotopic mass. </note>
In the param file it is also possible to define, taxonomy, database
and file locations etc? Do I need to pay any attention to this or may
I just select the mzXML file, the param file, and fasta database
through Petunia and hit the search button? :)
/Sincerely Marcus
On 11 Okt, 20:30, Luis Mendoza <[email protected]>
wrote:
> 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]>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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>
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