Hi Luis, Have there been any changes to Libra or planned future changes in upcoming releases to enable one to select/deselect which peptides are used for protein-level quantitation? In some experimental cases, I don't expect to see signal in one or more of the TMT channels (vehicle control, sham treatment etc) so these peptides don't get used for protein level quant. Any plans for supporting EASI-TAG quantification?
Best, Adam On Tuesday, November 21, 2017 at 5:06:55 PM UTC-6, Luis wrote: > > 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] <javascript:>> > 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/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. >>>> 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] <javascript:>. >> To post to this group, send email to [email protected] >> <javascript:>. >> 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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