Thanks Eric and Luis! Turns out I was using the wrong/older syntax in the condition file (targetMS level = “3”). The quantification works now. Thanks for your help!!!
Cheers, Yasir > On Sep 8, 2022, at 6:15 PM, 'Luis Mendoza' via spctools-discuss > <[email protected]> wrote: > > Hi Yasir, > Is your data Thermo SPS where the reporter ions are in MS3? If so, please > check the relevant box when producing the condition file: > > <image.png> > > > As Eric noted, it does not seem like your MS2 spectra contain reporter ion > signals, at least from the one spectrum you posted. Are there others where > this is not the case? > > Cheers, > --Luis > > > On Thu, Sep 8, 2022 at 2:28 PM 'Eric Deutsch' via spctools-discuss > <[email protected] > <mailto:[email protected]>> wrote: > I’m thinking that ion trap spectra are usually unsuitable for TMT: > > > <image001.png> > > > I think you would want to acquire fragmentation spectra with Orbitrap HCD? > > > > From: [email protected] > <mailto:[email protected]> <[email protected] > <mailto:[email protected]>> On Behalf Of Yasir Ahmed > Sent: Thursday, September 8, 2022 2:19 PM > To: [email protected] > <mailto:[email protected]> > Subject: Re: [spctools-discuss] Libra quantification on TMT 16-plex returns > zeros only > > > Hi Luis, > > > Just to follow up: I found a handful of cases where one of the channels has > super high quantification and observable reporter ions (see screenshots > below), albeit it should be way more than this, I believe. I’ll keep digging, > but let me know if you have any other ideas. > > > Again, thanks for you suggestions. > > > Cheers, > > Yasir > > > <image002.png> > > > <image003.png> > > > <image004.png> > > > > > On Sep 8, 2022, at 5:07 PM, Yasir Ahmed <[email protected] > <mailto:[email protected]>> wrote: > > > Hi Luis, > > > Thanks a lot for the response. > > > Can you try re-running Libra without the centroiding option? (i.e. set > type="0") Or are your mzML data not centroided? > > > I just tried that, but no difference in outcome. > > > > > Another thing to try: are you able to open the PeptideProphet + Libra results > in the PepXMLViewer? You should be able to see if there are a significant > number of quantified PSMs -- you should also be able to examine a few > individual spectra and zoom in on the reporter ion signals. > > > I’m able to look at a few individual spectra, and I think they all look > mostly good—but frankly I’m not too sure how to evaluate their suitability > (see example screenshots below). > > > > > Lastly, what version of TPP are you running? > > > 6.1.0 > > > Thanks again! > > Yasir > > > <Screen Shot 2022-09-08 at 5.00.50 PM.png> > > > <Screen Shot 2022-09-08 at 5.01.42 PM.png> > > > > > > > Cheers, > > --Luis > > > > On Thu, Sep 8, 2022 at 12:02 PM Yasir Ahmed <[email protected] > <mailto:[email protected]>> wrote: > > Greetings, > > > I'm having difficulty performing TMT quantification using Libra on a 16-plex > dataset. Basically, the "quantification.tsv" file that Libra outputs only > contains zeros for both peptides and proteins. The Comet search runs just > fine. I'm at a loss for where things are going wrong. The Mass Spec facility > used ProteomeDiscoverer for the quantification, and it worked fine so the > issue is not the raw data. > > > My TPP run repository can be found here > <https://github.com/YazBraimah/TPP_TMT_proteomics_pipeline>, and the > "condition.xml" file for Libra—along with the Comet params file—is in the > "params" folder. The commands used to run the TPP processes can be found in > the file called "Snakefile". I'm happy to provide any additional information > needed to help troubleshoot this. Any pointers/advise would be greatly > appreciated. > > > Cheers, > > Yasir > > > -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To unsubscribe from this group and stop receiving emails from it, send an > email to [email protected] > <mailto:[email protected]>. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/7cb42706-53ae-4c94-acd2-ea0293fea8b2n%40googlegroups.com > > <https://groups.google.com/d/msgid/spctools-discuss/7cb42706-53ae-4c94-acd2-ea0293fea8b2n%40googlegroups.com?utm_medium=email&utm_source=footer>. > > > -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To unsubscribe from this group and stop receiving emails from it, send an > email to [email protected] > <mailto:[email protected]>. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/CACyS9bo%3DQxLBhn3H%2B7cg9pTKXu1Yp1bTLwb%2BfF%3DR4y%2BujoF%3DxQ%40mail.gmail.com > > <https://groups.google.com/d/msgid/spctools-discuss/CACyS9bo%3DQxLBhn3H%2B7cg9pTKXu1Yp1bTLwb%2BfF%3DR4y%2BujoF%3DxQ%40mail.gmail.com?utm_medium=email&utm_source=footer>. > > > > -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To unsubscribe from this group and stop receiving emails from it, send an > email to [email protected] > <mailto:[email protected]>. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/5C533052-EF37-4516-AE3D-D90A364E5E68%40gmail.com > > <https://groups.google.com/d/msgid/spctools-discuss/5C533052-EF37-4516-AE3D-D90A364E5E68%40gmail.com?utm_medium=email&utm_source=footer>. > > > -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To unsubscribe from this group and stop receiving emails from it, send an > email to [email protected] > <mailto:[email protected]>. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/aad015ab22f19ee0cea8cd2cdc24627d%40mail.gmail.com > > <https://groups.google.com/d/msgid/spctools-discuss/aad015ab22f19ee0cea8cd2cdc24627d%40mail.gmail.com?utm_medium=email&utm_source=footer>. > > -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To unsubscribe from this group and stop receiving emails from it, send an > email to [email protected] > <mailto:[email protected]>. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/CACyS9boObJ2nFUNDd_1K%2BMghDJy62GQEYu4R6zRn2CuS-7JwBQ%40mail.gmail.com > > <https://groups.google.com/d/msgid/spctools-discuss/CACyS9boObJ2nFUNDd_1K%2BMghDJy62GQEYu4R6zRn2CuS-7JwBQ%40mail.gmail.com?utm_medium=email&utm_source=footer>. -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To unsubscribe from this group and stop receiving emails from it, send an email to [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/7EBE121E-E4BC-495C-B288-EE9B84CA78F9%40gmail.com.
