In the scenario you describe, XPRESS would think that all of the identified peptides were "heavy" and quantify the corresponding light partner by always subtracting a mass from the identified peptide mass. So check your results to confirm whether or not this is the case; I'd have to think that half of the quantitation numbers are wrong (those where the +140 mod peptides were considered heavy).
The supported search method is to use static mods of 140 and a variable mod of 8. On Fri, Oct 22, 2010 at 2:38 PM, Mike <michael.gorm...@gmail.com> wrote: > In the MASCOT search, I specify a variable modification for the n- > terminus and lysine residue for both light and heavy tags. This > allows for the identification of unlabeled peptides. In this > framework, no variable mods is unlabeled, whereas variable mod with an > addition of 140 Da is light and variable mod with an addition of 148 > Da is heavy. Perhaps this is discrepancy that is causing me problems > with ASAP ratio. Should the MASCOT search be formatted such that the > light tag is a fixed modification and the difference between heavy and > light is a variable modification? > > On Oct 22, 5:26 pm, Jimmy Eng <jke...@gmail.com> 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 <michael.gorm...@gmail.com> 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 <jke...@gmail.com> 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 spctools-disc...@googlegroups.com. >> > To unsubscribe from this group, send email to >> > spctools-discuss+unsubscr...@googlegroups.com. >> > For more options, visit this group >> > athttp://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. > To post to this group, send email to spctools-disc...@googlegroups.com. > To unsubscribe from this group, send email to > spctools-discuss+unsubscr...@googlegroups.com. > 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. 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