Hi Jimmy, Many thanks for the XPress changes. The 13C quantitation options work fine -- the software does exactly what it should. However, the results in our experiments are not satisfying, but this seems to be an intrinsic problem of the labeling and not of the software (simply too many carbons per peptide resulting in a high fraction in not 100% heavy peptides). For accurate quantitation it would be necessary to quantify over the whole isotopic envelope, but I think neither XPRess nor ASAPRatio can do this, and it is probably not easy to implement. So I will stick with N15 where possible.
> There was mention of 15N support in ASAPRatio in the 4.2 announcement. > After doing more snooping, the following check-in added support for > initializing AA masses via the command line that states it > facilitatesmetaboliclabeling:http://sashimi.svn.sourceforge.net/viewvc/sashimi?view=rev&revision=3969 I will have a look into this but for the moment XPress seems to be the easier option. > Metaboliclabeling is just not common for us (we've never had to process > any such data ourselves) so it's not well supported. So anything you > can contribute back to better support this would be welcome. Feel free > to send me the bug fix unless another developer here is interested in > taking care of it. It's on the way. > > I would hope that the static AA modifications that one would implement > for a heavy search would correctly make it back into the pep.xml file > and conversely passed correctly to the spectrum viewer. But w/o > experience with that workflow and zero experience pushing Mascots > results through the TPP, I wouldn't be able to guess where the problems > might lie and how easy the fixes might be. At the moment I do not see how metabolic labeling could be correctly represented in the current version of the pepXML format at all. I think static modifiactions of all AA might not be sufficient as they could lead to conflicts with additional variable modifications. But maybe someone who is really proficient in the pepXML format could give some hints here? Thanks again Matthias > > Good luck! > > - Jimmy > > [email protected] wrote: > > Hi Jimmy, > > > thanks for the quick reply. > > >>> 1.) Is it possible to use 13C labeling with either XPress or > >>> ASAPRatio? I've found only options for 15N, so is there any chance to > >>> process 13C-labeled samples? > >> You can't analyze 13C labeling now with Xpress. I don't see 13C (nor > >> 15N) support mentioned in the ASAPRatioPeptideParser usage statement so > >> there's a decent chance you can't use ASAPRatio right now for 13C > >> analysis either. > > > Okay, now I've also seen that 15N support is not mentioned in the > > ASAPRatio usage statement. I was quite sure that I have read about 15N > > support for ASAPRatio in the announcement of TPP4.2. Perhaps I was > > just fantasizing. > > >> It was easy to extend the 15N option in Xpress to 13C labeling which I > >> just did with the source files I have checked out. But it might take > >> awhile before these changes make it out. I need to check in the mods, > >> we'll need a small TPP GUI update, and the next TPP release will need to > >> be released. > > > Oh, this would be great if your modifications were available. For the > > moment I would also be happy if you could just check in the files so I > > could get them and compile them here (or, if you don't mind, even mail > > them to me. I have some people here urgently waiting for some > > results...). I would also do some beta- (or alpha-) testing of the new > > feature ;-) > > >>> 2.) How to perform the searches (Mascot in my case)? As far as I > >>> understood I have to search each sample twice - once for the light and > >>> once for the heavy peptides. That means to modify the masses and > >>> modification files on the Mascot server for every heavy search, so it > >>> is quite uncomfortable, especially if the Mascot server is shared with > >>> a lot of other people... With version 2.2, Mascot offers an option for > >>>metaboliclabeling, resulting in the identification of light and heavy > >>> peptides in one run. Is it possible to process such a combined result > >>> file through the TPP? > >> For current Xpress analysis ofmetaboliclabeled samples, the searches > >> do need to be performed separately. > > >> I don't know how the modification specification in pepXML handles > >>metaboliclabeling and if the Mascot to pepXML converter can correctly > >> translate combined light/heavymetabolicsearch results to pep.xml > >> files. It's going to take time and effort from some volunteer to make > >> it possible to process a combined result through the TPP. > > > Okay, that's what I expected. After doing some seperated searches now > > I have the impression that the pepXML does not handlemetabolic > > labeling at all. For heavy peptides, the Spectrum view (plot-msms) > > shows complete nonsense. Also the mass of modifications on heavy > > peptides is wrong - methionine oxidation is recorded with a mass of > > 147 but should be 148 on an N15 peptide. Or have I done something > > wrong with the Mascot search or conversion? > > > Regarding Samy's mail, I have also found that the XPressDisplay shows > > only the light peptides. I think there is a bug in > > XPressCGIProteinDisplay.cxx - double use of the int k in two nested > > for loops. I can provide a fixed version if someone is interested ;-) > > > Best wishes > > - Matthias --~--~---------~--~----~------------~-------~--~----~ 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 -~----------~----~----~----~------~----~------~--~---
