Hello Alastair, Unless there is a mistake, I think the N15 mass string should be:
-mA72.03415R160.10111N116.04293D116.02694C161.0307E130.04259Q130.05858G58.02146H140.05891I114.08406L114.08406K130.09496M132.04049F148.06841P 98.05276S88.03203T102.04768W188.07931Y164.06333V100.06841 See the following post: https://groups.google.com/g/spctools-discuss/c/O-Ude_j6Nss/m/6Tl2Q-_hAAAJ However, I am not detecting correct results in your heavy 'H' search results. I would start your troubleshooting there, beginning with the heavy mass of proline. Sorry this has taken me some time to get to and keep me posted to your progress. Cheers, -David On Wed, Aug 26, 2020 at 2:49 AM 'Alastair Skeffington' via spctools-discuss <spctools-discuss@googlegroups.com> wrote: > Hi David, > > Did you manage to grab the data before the link expired? > > Thanks, > Alastair > > On Monday, 17 August 2020 at 21:23:55 UTC+2 Alastair Skeffington wrote: > >> Hi David, >> >> Ah sorry - my mistake. Here's a new link with a tarball including the >> mxXML files. >> >> https://we.tl/t-UnX74n4qod >> >> Many thanks! >> Alastair >> >> >> On Monday, 17 August 2020 20:51:54 UTC+2, David Shteynberg wrote: >> >>> Hello Alastair, >>> >>> I downloaded the file you shared with me, however, there were no mzML >>> files to match the pepXML files so I couldn't actually try the analysis. >>> I will make another attempt if you can provide the mass spec data. >>> >>> Thanks, >>> -David >>> >>> On Mon, Aug 17, 2020 at 11:21 AM 'Alastair Skeffington' via >>> spctools-discuss <spctools...@googlegroups.com> wrote: >>> >> Hi David, >>>> >>>> The link to the data has now expired, Let me know if you are still >>>> willing to have a look and I can send it again. >>>> >>>> Otherwise maybe you could briefly describe the process you would go >>>> through? >>>> >>>> Many thanks, >>>> Alastair >>>> >>>> On Thursday, 6 August 2020 22:17:00 UTC+2, Alastair Skeffington wrote: >>>>> >>>>> Hi David, >>>>> >>>>> Many thanks for your reply. >>>>> >>>>> So it any peptides with modifications will simply be ignored for >>>>> quantification and I can ignore the warning message? >>>>> >>>>> Yes - each search was either light or heavy as defined in the static >>>>> modifications. >>>>> >>>>> And the -r parameter is then the window to search in the RT dimension? >>>>> Because the command line option says 'range around precursor m/z to search >>>>> for peak' I assumed this was for the m/z dimension. I thought that the >>>>> shift of the peak would also mostly be in the m/z dimension - but I'm no >>>>> mass spectrometrist! >>>>> >>>>> I would be amazing if you had a moment to have a look at the data - >>>>> thanks very much for offering. I've put two example pairs of files here: >>>>> https://we.tl/t-cVDD1MVDOr >>>>> >>>>> For each sample there is a light 'L' version of the search results and >>>>> a heavy 'H' version. I've also included the search database (based on some >>>>> PacBio data some I'm afraid it's quite big with a lot of isoforms). >>>>> >>>>> Many thanks, >>>>> Alastair >>>>> >>>>> On Wednesday, 5 August 2020 20:37:16 UTC+2, David Shteynberg wrote: >>>>>> >>>>>> Hi Alastair, >>>>>> >>>>>> If your search results are either all heavy or all light (not >>>>>> variable mod searched) then you should also use option -S. >>>>>> >>>>>> 1). You cannot specify anything but single amino acids in this >>>>>> string. Your quantitation will be based on peptides without PTMs in this >>>>>> dataset. >>>>>> >>>>>> 2). -r8 is a MUCH too wide window to recover the MS1 signal in >>>>>> RTspace. The lower this number the more selective the tool is at >>>>>> isolating >>>>>> your target signal. With -r8 you will not be quantifying the correct >>>>>> signal, unless you have a very bare sample. >>>>>> >>>>>> If you are able to share this data I can try running it to help you >>>>>> optimize your settings. >>>>>> >>>>>> Thanks, >>>>>> -David >>>>>> >>>>>> On Wed, Aug 5, 2020 at 11:02 AM 'Alastair Skeffington' via >>>>>> spctools-discuss <spctools...@googlegroups.com> wrote: >>>>>> >>>>>>> Hello, >>>>>>> >>>>>>> I'm trying to run a 14N/15N labelling experiment through ASAPRatio. >>>>>>> I've been running the first steps like this - here for the results of a >>>>>>> database search with heavy masses: >>>>>>> >>>>>>> InteractParser sample_interact.pep.xml sample.pep.xml >>>>>>> >>>>>>> PeptideProphetParser sample_interact.pep.xml >>>>>>> >>>>>>> RefreshParser sample_interact.pep.xml >>>>>>> ./EhuxAllproteins_MCC_decoy.fasta >>>>>>> >>>>>>> ASAPRatioPeptideParser sample_interact.pep.xml >>>>>>> -lACDEFGHIKLMNPQRSTVWY -r8 >>>>>>> -mA72.0779R160.1857N116.1026D116.0874C104.1429E130.1140Q130.1292G58.0513H140.1393I114.1576L114.1576K130.1723M132.1961F148.1739P99.1152S89.0773T102.1038W188.0793Y164.0633V101.1311 >>>>>>> >>>>>>> At this point I get a warning: >>>>>>> >>>>>>> WARNING: Found more than one variable mod on 'M'. Please make sure >>>>>>> to specify a heavy mass for this residue >>>>>>> >>>>>>> So I have two questions: >>>>>>> >>>>>>> 1) How do I specify the heavy mass for oxidised methionine? Mox ? >>>>>>> And is phosphorylated serine coded Sp ? >>>>>>> >>>>>>> 2) I've used -r8 instead of the default 0.5. My reasoning is that a >>>>>>> medium sized heavy peptide could easily differ from the 14N counterpart >>>>>>> by >>>>>>> 16 Da. Assuming charge +2, then using a m/z range of 8. Does this sound >>>>>>> remotely sensible? >>>>>>> >>>>>>> Many thanks! >>>>>>> Alastair >>>>>>> >>>>>>> -- >>>>>>> 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...@googlegroups.com. >>>>>>> To view this discussion on the web visit >>>>>>> https://groups.google.com/d/msgid/spctools-discuss/4853fda5-cc2f-48e2-a9e2-2ae93225b288o%40googlegroups.com >>>>>>> <https://groups.google.com/d/msgid/spctools-discuss/4853fda5-cc2f-48e2-a9e2-2ae93225b288o%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...@googlegroups.com. >>>> >>> To view this discussion on the web visit >>>> https://groups.google.com/d/msgid/spctools-discuss/3f3e8d01-8fd7-4d40-acde-1b7147769108o%40googlegroups.com >>>> <https://groups.google.com/d/msgid/spctools-discuss/3f3e8d01-8fd7-4d40-acde-1b7147769108o%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/c46993a6-b014-4790-b6bd-5afa5810e550n%40googlegroups.com > <https://groups.google.com/d/msgid/spctools-discuss/c46993a6-b014-4790-b6bd-5afa5810e550n%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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