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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