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 On Wed, Oct 12, 2011 at 11:58 AM, Marcus Sjödin <[email protected]>wrote: > 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 > > > > > -- > > > 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.- Dölj citerad > text - > > > > - Visa citerad text - > > -- > 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. > > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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