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.
