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 <[email protected]> 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 <[email protected]> >>>> 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 [email protected]. >>>>> 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 [email protected]. >>> >> 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 [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/ff3c5116-ca46-4aec-9ee4-d07d574db90an%40googlegroups.com.
