Hello Shagun, Perhaps I am misunderstand here. As far as I know, TMT labels are isobaric and are quantified in the fragment ion spectra. Any peptide quantified would have to have the same precursor mass across all TMT labels. Presumably there are not that many peptides that are identical between yeast and human. The difference in your ratios will only be observable in the conserved peptides between human and yeast. Do you have examples of specific spectra we can consider that work with this approach? I was able to run the TPP tools including Libra on this data.
Cheers, David On Sat, Mar 26, 2022, 8:11 PM Shagun Gupta <sg2...@cornell.edu> wrote: > Hi David > > So I reran with the changed parameter and the issue still seems to > persist. I can share the updated results if you'd like as well? Also just > confirming libra1 - 126, libra2 = 127N and so forth for a TMT10plex for > example, since the issue is much aggravated in 2 out of three possible > comparisons. To explain the setup more, yeast proteins are spiked with > mammalian proteins such that yeast proteins are 10:4:1 (three replicates > each) with mammalian proteins being (1:1:1) for the same. > > Shagun > > On Friday, March 25, 2022 at 6:02:55 PM UTC-4 Shagun Gupta wrote: > >> Hi David >> >> Will do so, thank you! That makes a lot of sense. I have also added the >> mzXML files but this might be the cause of the discrepancy I see! >> >> Thanks >> Shagun >> >> On Friday, March 25, 2022 at 4:42:46 PM UTC-4 David Shteynberg wrote: >> >>> Hello Shagun, >>> >>> I noticed in your condition file you are using TMT10 with the following >>> masses: >>> <reagent mz="126.127726" /> >>> <reagent mz="127.124761" /> >>> <reagent mz="127.131081" /> >>> <reagent mz="128.128116" /> >>> <reagent mz="128.134436" /> >>> <reagent mz="129.131471" /> >>> <reagent mz="129.137790" /> >>> <reagent mz="130.134825" /> >>> <reagent mz="130.141145" /> >>> <reagent mz="131.138180" /> >>> >>> >>> The difference between neighboring channels is <0.01 at the lowest and >>> yet you are using tolerance of 0.2: >>> >>> <massTolerance value="0.2" /> >>> >>> >>> I think the appropriate mass tolerance for this type of labeling should >>> be ~0.001. >>> >>> Does that make sense? Please try running Libra with the mass tolerance >>> appropriate for this type of label. >>> >>> Cheers, >>> -David >>> >>> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta <sg2...@cornell.edu> >>> wrote: >>> >>>> Hi David >>>> >>>> Could you suggest a good email to reach you with? I can share the >>>> pep.xml's and Libra condition file that way? >>>> >>>> -Shagun >>>> >>>> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote: >>>> >>>>> Hello Shagun, >>>>> >>>>> Thank you for your email and interest in the TPP. I have recently >>>>> been comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that >>>>> produced by TPP's Libra. As far as I can tell, when I run and compare the >>>>> quantities (intensities) they are mostly the same between Libra (without >>>>> isotopic impurity correction and 0 pseudocounts) and the >>>>> ProteomeDiscoverer/Byonic. Execution of Libra is defined in the >>>>> conditions.xml file that has to be defined for each Libra run. I would be >>>>> happy to take a look at your data and analysis to see if it can be placed >>>>> on the right path for the Libra analysis to work. Please post your >>>>> results >>>>> somewhere I can download and test. >>>>> >>>>> Cheers, >>>>> -David >>>>> >>>>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta <sg2...@cornell.edu> >>>>> wrote: >>>>> >>>>>> Hello >>>>>> >>>>>> Our lab has been trying to use Libra to quantify MS2 (Ion-trap) >>>>>> identification and MS3-based quantification of TMT datasets and we were >>>>>> trying to benchmark with an artificial yeast and mammalian spiked dataset >>>>>> with known fold-change (FC) values. However we observe drastically >>>>>> different values than expected, something we don't observe with other >>>>>> search engines for the same dataset. >>>>>> >>>>>> Has this issue been encountered before/ is there something obviously >>>>>> wrong when running with TPP that might cause this? Happy to provide >>>>>> further >>>>>> details on the dataset and parameters used to run with (most of them >>>>>> default apart from additions like static modification for TMT and >>>>>> specification of MS3 for quantification among others). >>>>>> >>>>>> Thank you, >>>>>> Shagun >>>>>> >>>>>> -- >>>>>> 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 spctools-discu...@googlegroups.com. >>>>>> To view this discussion on the web visit >>>>>> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com >>>>>> <https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%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 spctools-discu...@googlegroups.com. >>>> >>> To view this discussion on the web visit >>>> https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%40googlegroups.com >>>> <https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%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 spctools-discuss+unsubscr...@googlegroups.com. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/ff3c5116-ca46-4aec-9ee4-d07d574db90an%40googlegroups.com > <https://groups.google.com/d/msgid/spctools-discuss/ff3c5116-ca46-4aec-9ee4-d07d574db90an%40googlegroups.com?utm_medium=email&utm_source=footer> > . > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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