iTRAQ data through xtandem! using TPP
380 views
Skip to first unread message
Marcus Sjödin
Sep 27, 2011, 2:33:28 AM9/27/11
to spctools...@googlegroups.com
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
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
Luis Mendoza
Oct 11, 2011, 2:30:27 PM10/11/11
to spctools...@googlegroups.com
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
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
--
You received this message because you are subscribed to the Google Groups "spctools-discuss" group.
To view this discussion on the web visit https://groups.google.com/d/msg/spctools-discuss/-/d0rlZFFdG6cJ.
To post to this group, send email to spctools...@googlegroups.com.
To unsubscribe from this group, send email to spctools-discu...@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en.
Marcus Sjödin
Oct 12, 2011, 2:58:14 PM10/12/11
to spctools-discuss
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 <Luis.Mend...@systemsbiology.org>
wrote:
> >http://groups.google.com/group/spctools-discuss?hl=en.- Dölj citerad text -
>
> - Visa citerad text -
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 <Luis.Mend...@systemsbiology.org>
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
> <marcus.sjodi...@gmail.com>wrote:
> 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
>
> - Visa citerad text -
Luis Mendoza
Oct 13, 2011, 3:54:00 PM10/13/11
to spctools...@googlegroups.com
Hi Marcus,
Since the iTRAQ reporter ions are not part of the fragment ions from the peptide, it is better to leave them off of the window for identification - the software downstream takes care of quantitating based on those peaks. So, leaving the minimum at 125.0 is fine; setting it to 100.0 might confuse the algorithm.
As for the mass tolerances, we typically recommend leaving them on the large side, and using the accurate mass model in PeptideProphet to help discriminate the correct from incorrect results. You can always try out a few different approaches to see what gives you best results.
Cheers,
--Luis
Since the iTRAQ reporter ions are not part of the fragment ions from the peptide, it is better to leave them off of the window for identification - the software downstream takes care of quantitating based on those peaks. So, leaving the minimum at 125.0 is fine; setting it to 100.0 might confuse the algorithm.
As for the mass tolerances, we typically recommend leaving them on the large side, and using the accurate mass model in PeptideProphet to help discriminate the correct from incorrect results. You can always try out a few different approaches to see what gives you best results.
Cheers,
--Luis
Alberto Labarga
Sep 22, 2014, 1:55:53 PM9/22/14
to spctools...@googlegroups.com
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>
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
Frédéric DELOLME
Oct 8, 2014, 9:01:45 AM10/8/14
to spctools...@googlegroups.com
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
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
To unsubscribe from this group and stop receiving emails from it, send an email to spctools-discu...@googlegroups.com.
To post to this group, send email to spctools...@googlegroups.com.
Visit this group at http://groups.google.com/group/spctools-discuss.
For more options, visit https://groups.google.com/d/optout.
--
Frédéric DELOLME
Institut de Biologie et Chimie des Protéines - CCMP - Spectrométrie de masse
7, passage du Vercors - 69367 LYON Cedex 07 - FRANCE
Tél : +33 (0)4 72 72 26 93 - Fax : +33 (0)4 72 72 26 04
Reply all
Reply to author
Forward
0 new messages