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 > > -- > 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 [email protected]. > To unsubscribe from this group, send email to > [email protected]. > For more options, visit this group at > http://groups.google.com/group/spctools-discuss?hl=en. > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To post to this group, send email to [email protected]. To unsubscribe from this group, send email to [email protected]. For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en.
