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 <[email protected]> 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 <[email protected]> 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 [email protected]. >>>>>> 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 [email protected]. >>> >> 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. 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