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] <javascript:>> 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] <javascript:>. >> 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/93b97924-4a1e-4ea2-8e67-547dadde35d6o%40googlegroups.com.
