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] <javascript:>> 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] <javascript:>. >> 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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