Hi Adam, Do those psm's have a reported Libra result in the PepXMLViewer? Do all of these peptides come from a single run? Or is this a combined analysis of multiple runs?
Also might want to try using 5.1 (if not already doing so) in case there was a bug that got fixed. Cheers, --Luis On Tue, Nov 21, 2017 at 2:37 PM, Adam R <[email protected]> wrote: > Hi Luis, yes I checked the spectra and I can find the TMT reporter peaks. > Regarding the prot.xml file there is not an easy way for me to share it > externally...are there any other metrics that are used to discriminate > between which peptides are used for quantitation at the protein level with > TMT? > > Best, > > Adam > > On Friday, November 17, 2017 at 8:52:57 PM UTC-6, Luis wrote: >> >> Hi Adam, >> >> Yes, that is the column where the weight is found when displaying >> peptides. One last item: can you check if all of those spectra that >> correspond to those peptides contain TMT peaks? >> >> If you are able to share your protXML file, I could have a closer look. >> >> Cheers, >> --Luis >> >> >> On Tue, Nov 14, 2017 at 3:06 PM, Adam R <[email protected]> wrote: >> >>> Hi Luis, >>> >>> Are you referring to the "%Coverage/Weight" column that is in the >>> prot.xml file when you say "peptide be matched to the protein via weight > >>> 0.5" ? I selected the protein in the prot.xml to bring up the view with all >>> of the peptides ID'd. All the peptides that I see in this window (11 of >>> them) have 1.00 right next to the yellow bar under this column >>> "%Coverage/weight" Is there another weight value written that I am missing? >>> >>> Best, >>> >>> Adam >>> >>> >>> On Friday, November 10, 2017 at 6:04:57 PM UTC-6, Luis wrote: >>>> >>>> Hi Adam, >>>> >>>> A further requirement is that the peptide be matched to the protein via >>>> a weight > 0.5 (i.e. used in the calculation of the protein probability as >>>> assigned by ProteinProphet). Let us know if this is not the case and we >>>> can delve into it further. >>>> >>>> Cheers, >>>> --Luis >>>> >>>> >>>> On Fri, Nov 10, 2017 at 2:05 PM, Adam R <[email protected]> wrote: >>>> >>>>> Hi Luis, >>>>> >>>>> Yes, about one quarter of them have prob <0.5 but for the other ones >>>>> with prob and iprob >0.5 I'm not sure why I'm not seeing this in the >>>>> quantitation.tsv... >>>>> >>>>> Best, >>>>> >>>>> Adam >>>>> >>>>> On Friday, November 10, 2017 at 3:48:16 PM UTC-6, Luis wrote: >>>>>> >>>>>> Hi Adam, >>>>>> >>>>>> Hard to tell exactly without looking at your file. One thing to note >>>>>> is that Libra will only use psm's with PeptideProphet probabilities > >>>>>> 0.5; >>>>>> it does not take into account iProphet probabilities (yet). Maybe this >>>>>> explains some of the ones that are missing? >>>>>> >>>>>> Cheers, >>>>>> --Luis >>>>>> >>>>>> On Fri, Nov 10, 2017 at 1:14 PM, Adam R <[email protected]> wrote: >>>>>> >>>>>>> Hi Luis, >>>>>>> >>>>>>> I just took a look at the quantitation.tsv file. Interestingly >>>>>>> enough for the protein I am looking at there is only one peptide listed >>>>>>> in >>>>>>> this table, however, when looking at the ipro.pep.xml file for peptides >>>>>>> of >>>>>>> this protein I have eleven of them, all with ipro > 0.99 and some with >>>>>>> intensities of 0 in the competitive inhibitor-treated channel...not sure >>>>>>> what to make of this. Other proteins are listed with peptides and have >>>>>>> the >>>>>>> yes/no indicator for "kept?" and show intensities, in some cases zero... >>>>>>> Are there other parameters that are preventing these peptides from >>>>>>> showing >>>>>>> up in the quantitation.tsv file? >>>>>>> >>>>>>> Best, >>>>>>> >>>>>>> Adam >>>>>>> >>>>>>> On Friday, November 10, 2017 at 2:58:08 PM UTC-6, Luis wrote: >>>>>>>> >>>>>>>> >>>>>>>> I forgot to add that once the protein quantitation is done, Libra >>>>>>>> will also produce a "quantitation.tsv" file that you can use >>>>>>>> downstream for >>>>>>>> re-evaluating protein ratios with any statistical(s) test you wish -- >>>>>>>> say >>>>>>>> in R, Excel, etc. The file structure is fairly straightforward: >>>>>>>> protein >>>>>>>> ratios and errors, followed by those of its peptides, including >>>>>>>> adjusted >>>>>>>> intensities and a kept flag (used/not used in quant). >>>>>>>> >>>>>>>> ---Luis >>>>>>>> >>>>>>>> >>>>>>>> On Fri, Nov 10, 2017 at 12:49 PM, Adam R <[email protected]> wrote: >>>>>>>> >>>>>>>>> Thanks Luis for the response! >>>>>>>>> >>>>>>>>> Adam >>>>>>>>> >>>>>>>>> On Friday, November 10, 2017 at 2:30:33 PM UTC-6, Luis wrote: >>>>>>>>>> >>>>>>>>>> Hi Adam, >>>>>>>>>> >>>>>>>>>> We have plans to add some of these features to Libra as well as >>>>>>>>>> others, but none are ready at the moment. >>>>>>>>>> >>>>>>>>>> One thing you could try is to select to normalize against the sum >>>>>>>>>> of the reagent profiles (instead of selecting a channel to quantify >>>>>>>>>> against) -- this should give you a value for all channels, even when >>>>>>>>>> some >>>>>>>>>> are missing. >>>>>>>>>> >>>>>>>>>> Cheers, >>>>>>>>>> --Luis >>>>>>>>>> >>>>>>>>>> >>>>>>>>>> On Fri, Nov 10, 2017 at 11:44 AM, Adam R <[email protected]> >>>>>>>>>> wrote: >>>>>>>>>> >>>>>>>>>>> Is there a way to retain quantified peptides for the protein >>>>>>>>>>> quant with Libra? I understand that Libra discards peptides without >>>>>>>>>>> all >>>>>>>>>>> channels...what about in instances where the you expect there to be >>>>>>>>>>> little >>>>>>>>>>> or no intensity? I have data from pulldowns where we don't expect >>>>>>>>>>> to see >>>>>>>>>>> much of the protein in the presence of a competitive inhibitor, so >>>>>>>>>>> in this >>>>>>>>>>> case it would be of use to retain such quants where the intensity >>>>>>>>>>> is zero >>>>>>>>>>> in the presence of inhibitor but several thousands in the pulldown >>>>>>>>>>> channel...Is there a way to toggle on/off the peptides that are >>>>>>>>>>> used for >>>>>>>>>>> the protein quant in Libra? I'm thinking along the lines like in >>>>>>>>>>> Xpress >>>>>>>>>>> where you can toggle whether or not a peptide is used for the >>>>>>>>>>> protein >>>>>>>>>>> quant. Is there also a way to modify the number of standard >>>>>>>>>>> deviations that >>>>>>>>>>> are used to discard a peptide from being used in the protein quant? >>>>>>>>>>> >>>>>>>>>>> Best, >>>>>>>>>>> >>>>>>>>>>> Adam >>>>>>>>>>> >>>>>>>>>>> -- >>>>>>>>>>> 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 post to this group, send email to >>>>>>>>>>> [email protected]. >>>>>>>>>>> Visit this group at https://groups.google.com/grou >>>>>>>>>>> p/spctools-discuss. >>>>>>>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>>>>>>> >>>>>>>>>> >>>>>>>>>> -- >>>>>>>>> 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 post to this group, send email to [email protected]. >>>>>>>>> Visit this group at https://groups.google.com/grou >>>>>>>>> p/spctools-discuss. >>>>>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>>>>> >>>>>>>> >>>>>>>> -- >>>>>>> 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 post to this group, send email to [email protected]. >>>>>>> Visit this group at https://groups.google.com/group/spctools-discuss >>>>>>> . >>>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>>> >>>>>> >>>>>> -- >>>>> 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 post to this group, send email to [email protected]. >>>>> Visit this group at https://groups.google.com/group/spctools-discuss. >>>>> For more options, visit https://groups.google.com/d/optout. >>>>> >>>> >>>> -- >>> 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 post to this group, send email to [email protected]. >>> Visit this group at https://groups.google.com/group/spctools-discuss. >>> For more options, visit https://groups.google.com/d/optout. >>> >> >> -- > 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 post to this group, send email to [email protected]. > Visit this group at https://groups.google.com/group/spctools-discuss. > For more options, visit https://groups.google.com/d/optout. > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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