Hi DT, I'm trying to use dimethylation labelled samples with TPP but I think I might not be specifying the modifications correctly. Is there any chance that you could post the relevant sections from your search params file (preferably X!Tandem if you're using this)?
Many thanks, Ben On Mar 19, 11:10 am, dctrud <[email protected]> wrote: > Bernard, > > Sorry for the slow reply. ASAPRatio will know which label is heaviest, > it shouldn't matter which way around the searches are. > > With regard to the questions r.e.dimethylfalse positives, the > procedure we are using this for involves labeling for quantitation > where all lysines should be dimethylated / heavy dimethylated. I > probably wrongly assumed you were doing this labeling, whereas I guess > you are not forcing complete dimethylation of lysines? What exactly is > your experiment? What biological changes are you trying to measure? > > DT > > On 11 Mar, 17:59, Bernt <[email protected]> wrote: > > > > > Hi DT, > > > Really grateful for your advice. I get what you mean, I have 2 more > > concerns: can we change which form to be the numerator and which form > > to be the denominator? When we specify -A-lnK-S, would the ratio be > > Search1 / Search 2 or Search 2 / Search 1? Do you get Peptide Prophet > > probabilities when you place a static mod on Lys? > > > Another interesting point for discussion in methylation: would you be > > able to distinguishdimethylLys from Arginine? Would placing static > > mod ofdimethylon Lys give you many false positives which might be > > just Arginines instead? How do you manage them? > > > Bernard > > > P.S: Has anybody come across methylated tryptophan W? > > > On Mar 11, 10:00 pm, dctrud <[email protected]> wrote: > > > > Bernard, > > > > We've successfully used ASAPRatio for heavydimethyllabeled > > > quantitation using the following workflow, which might help you get to > > > where you want: > > > > * Run *two* searches, with the light and heavy methyl n-term and > > > lysine mods specified as *static* modifications, i.e. > > > > Search 1:Dimethyl(K) +Dimethyl(n-term) as static mods. > > > Search 2:Dimethyl:2H4 (K) +Dimethyl:2H4 (n-term) as static mods. > > > > * Run xinteract on the resulting pair of search results using the > > > static mods option for ASAPRatio, i.e. > > > > -A-lnK-S-r0.05 > > > > There's no need to run XPRESS if you are only interested in ASAPRatio > > > results. > > > > DT > > > > On 11 Mar, 07:05, Bernt <[email protected]> wrote: > > > > > Dear all, > > > > > I have a question pertaining to ASAPRatio. > > > > > Can I actually get ASAPRatio to give me a ratio of Kmethylated / Kheavy > > > > +methylated (i.e. K142/K148)? If it can be done, how do I specify on > > > > the commandline different modified forms of Lys as the numerator and > > > > denominator of the ratio? Both modifications have been specified under > > > > variable mods in Xtandem. Does Xpress need to be specified everytime > > > > ASAPRatio is run, or can it be omitted? Can the following work? > > > > > xinteract -NinteractHeavyMethyl2.pep.xml -p0 -l6 -OA -A- > > > > lR170.11676K142.11061-F-r0.1-mK148.13061R180.12476 > > > > "*HeavyMethyl.tandem.pep.xml" > > > > > I also tried specifying static mod on Lys (i.e. 1...@k), and variable > > > > mod of 2...@k to represent the Heavy+Methyl form in Xtandem first, > > > > then : > > > > > xinteract -Ninteractstatic.pep.xml -p0 -l6 -OA -X-nR,10K,6 -A-lRK-F- > > > > r0.1-mK148.13061R180.12476 "*static.tandem.pep.xml" > > > > > But in this case, PeptideProphet's probabilities were all 0, > > > > presumably because there were very few significant peptide assignments > > > > due to the static mod specified on all Lys. Does anybody have any idea > > > > how I can get around this problem with ASAPRatio? > > > > > I look forward to any suggestions. Thanks in advance. > > > > > Cheers, > > > > Bernard -- 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]. For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en.
