Thanks for your help Jimmy and Ben. I solved this problem. I searched the files using MASCOT with the monoisotopic mass of the light tags set as Fixed modifications and the monoisotopic mass of the heavy tags set as Variable modifications. In the dat file, MASCOT lists the mass difference between the labeled residues. The mass difference was incorrectly calculated for the nTerminus. I created a script to manually go into the file and replace this value with the actual mass difference. The line I replaced reads "delta1=MassDiff,m- TRAQ-8(N-term)". Once I made these changes ASAP ratio was able to correctly identify the signals from the light and heavy tags.
On Oct 22, 5:39 pm, Ben Collins <[email protected]> wrote: > Hi Mike, > > Not sure if this is helpful or not but I've been able to make ASAPratio work > with binary chemical labeling (reductive dimethylation in my case) by doing > two separate searches with the light label specified as a static mod in one > and the heavy label specified as a static mod in the other. The outputs are > combined at the xinteract stage. Take a look at this post where I got some > advice on how to do this: > > http://groups.google.com/group/spctools-discuss/browse_thread/thread/... > > <http://groups.google.com/group/spctools-discuss/browse_thread/thread/...> > Cheers, > Ben > > > > On Fri, Oct 22, 2010 at 10:26 PM, Jimmy Eng <[email protected]> wrote: > > Determining light or heavy peptide is simple and shouldn't be a black > > box: No variable mods on the specified residues equals light peptide. > > Variable mods on all of the specified residues equals heavy peptide. > > If there's a mixture (in your case an unmodified + a modified lysine > > in the same peptide), then that peptide is ignored. > > > On Fri, Oct 22, 2010 at 2:20 PM, Mike <[email protected]> wrote: > > > Thanks for your prompt reply Jimmy. It is clear to me how XPRESS > > > calculates the mass difference between light and heavy labeled > > > peptides. As you describe, this is simply a matter of knowing the > > > number of labeled residues in the peptide and the associated mass > > > differences. It is unclear how the program identifies whether a > > > peptide is light labeled or heavy. For instance, let us say that I > > > have detected a doubly charged peptide at an m/z of 900. If this is > > > the light labeled peptide, I would expect to see the heavy labeled > > > peptide pair at an m/z of 904. If this is the heavy labeled peptide, > > > I expect to see the light labeled pair at an m/z of 896. I can assume > > > that XPRESS obtains the information regarding whether a peptide is > > > light or heavy labeled based on which variable modifications match the > > > spectra in the MASCOT search. This aspect of the analysis is working > > > however, so I am content to keep this process in a black box. > > > > I suspect my problems with ASAP ratio come from discrepancies in the > > > way the Mascot search was done and what ASAP ratio expects to find in > > > the pepXML file. > > > > On Oct 22, 4:53 pm, Jimmy Eng <[email protected]> wrote: > > >> > Similarly, if > > >> > only the difference between light and heavy residues are entered for > > >> > XPRESS, how does this program know the mass of the light and heavy > > >> > tagged residues? > > > >> Regarding this question, XPRESS doesn't need to know the mass of the > > >> tagged residues as that information isn't necessary. Knowing the > > >> labeled residues which you specify, it determines if a peptide is the > > >> light or heavy form based on the modifications from the search because > > >> you have to run the search in a specific way. Peptides with no > > >> variable modifications on the specified residues are "light" and > > >> peptides with variable modifications on all of the specified residues > > >> are "heavy". The mass of the identified peptide is known and > > >> calculated from the database search. XPRESS just needs to get the > > >> mass of the paired peptide now. > > > >> Once it knows that a peptide is either light or heavy, it can > > >> calculate the m/z of the corresponding pair by counting the number of > > >> labeled residues in the peptide (which it knows 'cause you specified > > >> the labeled residues) and their mass difference (which you also > > >> specify). The # of labeled residues and mass difference gives the > > >> mass to either add or subtract from the identified peptide mass to > > >> calculate the mass of the other pair. Hope I explained this clearly > > >> and hopefully someone else will chime in on your ASAPRatio questions. > > > > -- > > > You received this message because you are subscribed to the Google Groups > > "spctools-discuss" group. > > > To post to this group, send email to [email protected]. > > > To unsubscribe from this group, send email to > > [email protected]<spctools-discuss%[email protected]> > > . > > > For more options, visit this group at > >http://groups.google.com/group/spctools-discuss?hl=en. > > > -- > > You received this message because you are subscribed to the Google Groups > > "spctools-discuss" group. > > To post to this group, send email to [email protected]. > > To unsubscribe from this group, send email to > > [email protected]<spctools-discuss%[email protected]> > > . > > For more options, visit this group at > >http://groups.google.com/group/spctools-discuss?hl=en.- Hide quoted text - > > - Show quoted text - -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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